CN1820020B - SARS-coronavirus virus-like particles and methods of use - Google Patents

Abstract

The present disclosure describes a system for making SARS-CoV-virus-like particles (SARS-CoV-VLPs) comprising one or more recombinant vectors which express the SARS-CoV E-protein, the SARS-CoV M-protein, and the SARS-CoV S-protein. Additionally, the present disclosure describes methods of inducing an immune response in a subject comprising administering to the subject a nucleic acid encoding the SARS-CoV E-protein, the SARS-CoV M-protein, and the SARS-CoV S-protein. Methods of inducing an immune response in a subject comprising administering to the subject SARS-CoV-VLPs are also disclosed.

Description

SARS-coronavirus virus-like particles and application method

Invention field

The present invention relates to biotechnology and field of medicaments.Particularly, the present invention relates to SARS-coronavirus sample particle, the method for producing these particulate methods and utilizing these particle induction of immunity reactions.

Background technology

The outburst of sars coronavirus (SARS-CoV) causes a large amount of death in the world wide recently, particularly in China, Canada and Vietnam.Lethality is generally caused by the respiratory insufficiency of carrying out property, and it takes place in up to whole SARS cases of about 10%.At present, do not have methods of treatment for SARS and do not reduce the means of the mortality ratio of disease-related connection therewith.In addition, do not prevent the means of SARS infection without any the means that weaken the symptom that is associated with this infection reliably yet.Consider the great effect of SARS and lack suitable therapeutic method, be starved of composition and/or the method for improving this disease effector.

Summary of the invention

Embodiments more of the present invention relate to expresses SARS-CoV embrane-associated protein M in vitro and in vivo in such as the cell of people's cell, the system of E and S is as recombinant plasmid, virus and prokaryotic organism.In some embodiments, SARS-CoV M, E and S albumen spontaneously form SARS-CoV-virus-like particle (SARS-CoV-VLPs).In these embodiments, SARS-CoV-VLPs can be by emiocytosis.

According to other embodiments of the present invention, present the SARS-CoV M of virus protein in their " natural " environment, E and the proteic cell inner expression of S and with form virus-like particle relatedly caused immunoreactive inducing.Thus, certain embodiments of the present invention relate to the experimenter of the danger that is tested and appraised development SARS and use one or more to described experimenter and can express SARS-CoV M, the genetic constructs of E and/or S polypeptide comes animal, as producing immunoreactive method in people and other Mammals.In some embodiments, one or more genetic constructs are expressed SARS-CoV M, E and S polypeptide, its spontaneous formation SARS-CoV-VLPs.In a preferred embodiment, intensive antibody response and intensive cytotoxic T lymphocyte (CTL) are replied all and are induced.In other embodiment, have only the wherein a kind of of antibody response or ctl response to be induced.

Embodiments more of the present invention relate to SARS-CoV-VLPs and produce these particulate methods.Other embodiment relates to animal, as people or other administration SARS-CoV-VLPs, thereby produces immunne response in animal body.

In some embodiments of the present invention, the VLPs of generation comprises the E albumen that is selected from SEQ ID NOs:2-5 or its part.In other embodiment, the VLPs of generation comprises the M albumen that is selected from SEQ IDNOs:6-9 or its part.Still in other embodiment, the VLPs of generation comprises the S albumen that is selected from SEQ ID NOs:10-13 or its part.The proteic part of E can comprise about at least 6 continuous amino acids, at least about 7 continuous amino acids, at least about 8 continuous amino acids, at least about 9 continuous amino acids, at least about 10 continuous amino acids, at least about 11 continuous amino acids, at least about 12 continuous amino acids, at least about 13 continuous amino acids, at least about 14 continuous amino acids, about at least 15 continuous amino acids, about at least 16 continuous amino acids, at least about 17 continuous amino acids, at least about 18 continuous amino acids, about at least 19 continuous amino acids, about at least 20 continuous amino acids, at least about 25 continuous amino acids, at least about 30 continuous amino acids, about at least 40 continuous amino acids, about at least 50 continuous amino acids, at least about 60 continuous amino acids, about at least 70 continuous amino acids or greater than 70 amino acid.The proteic part of M can comprise about at least 6 continuous amino acids, at least about 7 continuous amino acids, at least about 8 continuous amino acids, at least about 9 continuous amino acids, at least about 10 continuous amino acids, at least about 11 continuous amino acids, at least about 12 continuous amino acids, at least about 13 continuous amino acids, at least about 14 continuous amino acids, at least about 15 continuous amino acids, at least about 16 continuous amino acids, at least about 17 continuous amino acids, at least about 18 continuous amino acids, at least about 19 continuous amino acids, at least about 20 continuous amino acids, at least about 25 continuous amino acids, at least about 30 continuous amino acids, at least about 40 continuous amino acids, at least about 50 continuous amino acids, at least about 60 continuous amino acids, about at least 70 continuous amino acids, about at least 80 continuous amino acids, at least about 90 continuous amino acids, at least about 100 continuous amino acids, about at least 120 continuous amino acids, about at least 140 continuous amino acids, at least about 160 continuous amino acids, at least about 180 continuous amino acids, about at least 200 continuous amino acids, or greater than 200 continuous amino acids.The proteic part of S can comprise about at least 6 continuous amino acids, at least about 7 continuous amino acids, at least about 8 continuous amino acids, at least about 9 continuous amino acids, at least about 10 continuous amino acids, at least about 11 continuous amino acids, at least about 12 continuous amino acids, at least about 13 continuous amino acids, at least about 14 continuous amino acids, at least about 15 continuous amino acids, at least about 16 continuous amino acids, at least about 17 continuous amino acids, at least about 18 continuous amino acids, at least about 19 continuous amino acids, at least about 20 continuous amino acids, at least about 25 continuous amino acids, at least about 30 continuous amino acids, at least about 40 continuous amino acids, at least about 50 continuous amino acids, at least about 60 continuous amino acids, at least about 70 continuous amino acids, at least about 80 continuous amino acids, at least about 90 continuous amino acids, at least about 100 continuous amino acids, at least about 120 continuous amino acids, at least about 140 continuous amino acids, at least about 160 continuous amino acids, at least about 180 continuous amino acids, at least about 200 continuous amino acids, at least about 250 continuous amino acids, at least about 300 continuous amino acids, at least about 350 continuous amino acids, at least about 400 continuous amino acids, at least about 450 continuous amino acids, at least about 500 continuous amino acids, at least about 550 continuous amino acids, at least about 600 continuous amino acids, at least about 650 continuous amino acids, at least about 700 continuous amino acids, at least about 750 continuous amino acids, at least about 800 continuous amino acids, at least about 850 continuous amino acids, about at least 900 continuous amino acids, about at least 950 continuous amino acids, at least about 1000 continuous amino acids, at least about 1050 continuous amino acids, about at least 1100 continuous amino acids, about at least 1150 continuous amino acids, at least about 1200 continuous amino acids, about at least 1250 continuous amino acids or greater than 1250 continuous amino acids.

System and method as herein described can be used for weakening the symptom that SARS-CoV infects.

Others of the present invention are provided in the paragraph of following label:

1. system for preparing SARS-CoV virus-like particle (SARS-CoV-VLPs), it comprises one or more and expresses SARS-CoV E albumen, SARS-CoV M albumen and the proteic recombinant vectors of SARS-CoV S.

2. the system of claim 1, wherein said SARS-CoV E albumen, described SARS-CoV M albumen and described SARS-CoV S albumen are expressed by single recombinant vectors.

3. the system of claim 1, wherein said SARS-CoV E albumen, described SARS-CoV M albumen and described SARS-CoV S albumen are expressed by a plurality of recombinant vectorss.

4. the system of claim 1, wherein said one or more recombinant vectorss comprise plasmid.

5. the system of claim 1, wherein said one or more recombinant vectorss comprise recombinant virus.

6. the system of claim 5, wherein said recombinant virus is a Measles virus.

7. cell, it has been transformed into expresses SARS-CoV E albumen, SARS-CoV M albumen and SARS-CoV S albumen.

8. the cell of claim 7, wherein said cell is alive.

9. the cell of claim 8, wherein said cell is a bacterial cell.

10. the cell of claim 9, wherein said cell is its pathogenic bacterial cell that has been weakened.

11. the cell of claim 10, wherein said cell are Salmonellas (Salmonella) cells.

12. the method for an induction of immunity reaction comprises that using one or more to the experimenter expresses SARS-CoV E albumen, SARS-CoV M albumen and the proteic recombinant vectors of SARS-CoV S.

13. the method for claim 12, wherein said SARS-CoV E albumen, SARS-CoV M albumen and SARS-CoV S albumen are expressed by single recombinant vectors.

14. the method for claim 12, wherein said SARS-CoV E albumen, SARS-CoV M albumen and SARS-CoV S albumen are expressed by a plurality of recombinant vectorss.

15. the method for claim 12, wherein said one or more recombinant vectorss comprise plasmid.

16. the method for claim 12, wherein said one or more recombinant vectorss comprise virus.

17. the method for claim 12, wherein said one or more recombinant vectorss comprise prokaryotic vector.

18. the method for claim 12, wherein said experimenter is the people.

19. the method for claim 18, wherein said immunne response is a cellullar immunologic response.

20. the method for claim 18, wherein said immunne response is a humoral immunoresponse(HI).

21. the method for claim 18, wherein said immunne response are cellullar immunologic response and humoral immunoresponse(HI).

22. the method for an induce immune response in subject comprises to described experimenter and uses SARS-CoV-VLPs.

23. the method for an induce immune response in subject comprises to described experimenter and uses encoding SARS-CoV E albumen, SARS-CoV M albumen and the proteic nucleic acid of SARS-CoV S.

24. SARS-CoV-VLP.

25. the SARS-CoV-VLP of an isolating claim 24.

The accompanying drawing summary

Fig. 1 has described the diagram of coronavirus.Geneome RNA is wrapped up by nucleocapsid protein N.Membranin M, furcella (spike) Protein S and E albumen are embedded in the lipid bilayer.Several coronavirus also comprise the 4th kind of envelope protein, hemagglutinin esterase protein HE (not shown).

Fig. 2 shows the genome organization of coronavirus.Shown main open reading-frame (ORF) by the geneome RNA coding of MHV, HCV 229E, IBV and SARS-CoV.To encode and integrate envelope protein S, the gene of M and E is shown as closed box.

Fig. 3 describes coronavirus genetic expression.Expressed the mRNAs of common terminal (coterminal) nested sets.The unique zone that only whether is comprised in the mRNA of next less mRNA is activated in translation.To encode and integrate envelope protein S, the gene of M and E is shown as closed box.

Fig. 4 A has described from four SARS-CoV strain isolated: Tor2, Urbani, the comparison of the E Argine Monohydrochloride sequence among HKU-39849 and the CUHK-W1.

Fig. 4 B has described from four SARS-CoV strain isolated: Tor2, Urbani, the comparison of the M Argine Monohydrochloride sequence among HKU-39849 and the CUHK-W1.Protuberance (Highlighting) is presented at residue all inequality between each of four strain isolateds.

Fig. 4 C has described from four SARS-CoV strain isolated: Tor2, Urbani, the comparison of the S Argine Monohydrochloride sequence among HKU-39849 and the CUHK-W1.Protuberance (Highlighting) is presented at residue all inequality between each of four strain isolateds.

After Fig. 5 was described in transfection and is that promoter in eukaryote control carries the plasmid of correlation gene down, transfected tissue culture cells produced SARS-CoV albumen.

After Fig. 6 was described in and is used in promoter in eukaryote control and carries the recombinant virus infection of correlation gene down, tissue culture cells produced SARS-CoV albumen.

Fig. 7 is described in and adds the SARS-CoV S-that is carried under the eukaryotic promoter control, and behind the prokaryotic vector of M-and E-gene, tissue culture cells produces SARS-CoV albumen.

Fig. 8 is described in SARS-CoV S-, and after M-and the E-genetic expression, tissue culture cells produces SARS-CoV sample particle.With described particle release in the tissue culture supernatant liquor.

Fig. 9 illustrates the density gradient centrifugation of the intact virion that Triton-X100 of no use (nothing) handles and the result of the migration of dissolved albumen (Triton-X100) in saccharose gradient with chart.

Detailed Description Of The Invention

Aspects more of the present invention provide expresses sars coronavirus (SARS-CoV) M, the transmission carrier of the product of E and S gene. Described transmission carrier can be the carrier that is suitable for any type of this purpose. For example, carrier can be plasmid vector, viral vectors or prokaryotic vector. Described carrier produces in vivo SARS-CoV-virus-like particle (SARS-CoV-VLPs) and can comprise any as follows:

A. carry a kind of carrier of all three genes (M, E, and S), or

B. two kinds of carriers, a kind of combination of carrying any two aforementioned genes, another carries aforementioned gene one of them, or

C. three kinds of carriers, every kind is carried one of them of aforementioned gene.

SARS-CoV-sample particle is effective to there be not the related indication animal moderate stimulation of diseases or SARS immune response.

Can pass through irritation cell and/or immunity system acquisition for effective host defense of the infectious disease of coronavirus association. In one embodiment, system disclosed herein will produce efficient antibody and stimulate t cell response owing to the intracellular expression of viral antigen with by discharging natural envelope protein as virus-like particle (VLPs) component. In another embodiment, described antigen will in the mucous membrane site, produce and existence such as lung.

VLPs can be used for required host, such as induce immune response among the people. Although the immune response of adequately protecting property is desirable, will be appreciated that the immune response of adequately protecting property of tool is not useful yet. Therefore, this invention is intended to induce the immune response of adequately protecting property and the immune response of non-adequately protecting property.

In some embodiments of the present invention, use gene or the polypeptide preparation SARS-CoV-VLPs of sars coronavirus. For example, one embodiment of the invention are intended to application vector and express enough the SARS-CoV M that produces VLPs, E and S gene or its part. The example that is used for the carrier of this expression is to be suitable for encoding proteins at any carrier of the effective expression of suitable cell type. These carriers can include, but not limited to plasmid vector, viral vectors and prokaryotic vector. So-called " prokaryotic vector " refers to a kind of microorganism, and it comprises and has one or more one or more plasmids of one or more genes of the relevant particle of SARS-CoV-of coding. Use the suitable cell type of these carriers from people and other mammal.

In some embodiments of the present invention, use DNA is transferred to cell, such as the proper delivery system in people's cell. For example, in one embodiment, but intracutaneous transmits naked DNA. In another embodiment, transmission system can with all three gene deliveries in the pneumonocyte to induce the mucous membrane immunity in sites of infection. Use single plasmid, a plurality of plasmids or other can once shift other system of several genes, but such as protokaryon or viral gene delivery system transfer gene.

In some embodiments of the present invention, described SARS-CoV gene, M, E and S express from the carrier that offers tissue culture. In some embodiments, with described SARS-CoV M, E and S protein are combined with the VLPs that self-organizing culture cell discharges. In certain embodiments, express SARS-CoV-VLPs from carrier disclosed by the invention when expressing in vivo, induce body fluid and/or cellullar immunologic response.

In other embodiments of the present invention, separated the SARS-CoV-VLPs that originates from cell culture, it has been mixed with immunogene and is administered to body fluid and/or the cellullar immunologic response that the risky experimenter who is infected by SARS induces the experimenter thus.

The coronavirus classification

Coronavirus is the member of nidovirales, and nidovirales is the order (order) (Jerusalem, 1996) of determining the 10th the viral conference in the world. Described order is by coronaviridae (coronaviridae) and arteriviridae section, and positive chain RNA virus forms, and they are attributed to identical order is because they similitudes on genome organization replication strategy similar with them. Coronavirus is similar to the feature outward appearance name that the large spike protein by stretching out virosomal surface of corona solis causes with them under electron microscope.

Coronavirus infection comprises people's multiple mammal, mainly causes and breathes or the intestines infection. Example and their hosts separately of the coronavirus at least three unique antigen group that clusters have been provided in the table 1. The nearest sars coronavirus that studies show that may be the member of II type coronavirus group.

Table 1: coronavirus and their host

Group host title acronym disease

I mouse MHV MHV hepatitis/encephalitis/intestines

Ox bovine coronavirus BCV enteritis

Everybody HCoV-OC43 HCoV OV43 enteritis

Pig pig blood clotting encephalomyelitis HEV respiratory infections

(encephalomyelitis) virus

Rat rat coronavirus RCV respiratory infections

Turkey turkey coronavirus TCV respiratory infections

II dog canine coronavirus CCV enteritis

Cat cat Infectious peritonitis virus FIPV respiratory infections

Cat cat intestines coronavirus FECV enteritis

Everybody HCoV-229E HCoV 229E respiratory infections

Pig pig infectious diarrhea virus PEDV enteritis

The transferable gastroenteritis of pig

Swine disease poison TGEV enteritis

Turkey turkey coronavirus TCV respiratory infections

The infectious segmental bronchus virus of III chicken bird IBV respiratory infection

The acute respiration symptom hat SARS CoV respiratory infection that designated person is not serious

Shape virus

The coronavirus structure

Coronavirus is tunicary virus.Virosome is that the lipid bilayer of the multiform particle of 80-200nm and host cell origin is around the geneome RNA that is wrapped up by nucleocapsid protein.Evidence from early stage research shows that the packaged form of coronavirus nucleocapsid is spiral helicine (MacNaughton etc., 1978).Yet, the newer data presentation of appearance, more high-grade, nucleocapsid is packaged in the icosahedro form in the virosome (Risco etc., 1996).The diagrammatic form of coronavirus body is shown among Fig. 1.

Except that the nucleocapsid structure, coronavirus comprises other various structural polypeptides.For example, coronavirus comprises triple film M albumen (20-25kD) of striding, and it is the abundantest envelope protein.Another structural protein are spike proteins, S (180kD), and it forms the coating grain on virosomal surface.S is incorporated into coronavirus acceptor and inducing cell-cytogamy and virus-cytogamy and neutralizing antibody.Recently, find little envelope protein, E is the part (Liu and Inglis, 1991) of virosome.As if described E albumen be essential (Venemma etc., 1996) for the assembling of virus.Each of said structure characteristics described in Fig. 1 to some extent.

The coronavirus genome organization

The geneome RNA of coronavirus is contained 27-32kB.5 ' 2/3 the rdrp gene of genome encoding in two big overlapping open reading-frame (ORF)s.Structural protein S, M, E and N are by genomic 3 ' terminal coding (see figure 2).In addition, there is the several little open reading-frame (ORF) (ORFs) that intersperses among between the structure gene.Yet, do not know what whether and how ORFs expressed always, do not know the effect of these open reading-frame (ORF)s always yet.The supposition of some some effects that may play in viral pathogeny about these little ORFs has been arranged.

Coronavirus is duplicated and genetic expression

After the infection, thereby coronavirus RNA is translated the RNA polymerase that produces by the dependenc RNA of eclipsed open reading-frame (ORF) 1a and 1b coding.Latter ORF only in (1) ribosomal frameshift incident the back takes place to be expressed, and described frameshit incident takes place with the frequency of as many as about 30%.Because the coronavirus genome is straight polarity, strand RNA synthesize next step generation in replicative cycle.Strand RNA serves as the template of new positive strand genomic rna successively.Except 3 of the comfortable nested setses of all gene translations of rdrp gene '-terminal mRNAs altogether, it comprises unique zone and comprises one or more ORFs at the 5-end, described unique zone is not included among the next less mRNA.Usually, the coronavirus genome is included in and transcribes correlated series (TAS) element before each open reading-frame (ORF).Fig. 3 shows use HCV 229E typical coronavirus genome organization as an example.For HCV 229E, TAS is UCUAAACU (SEQ ID NO:1).

Coronavirus structural protein M, E and S that film is relevant

M protein

Described M albumen is the abundantest membranin in the coronavirus virus body.The C-end is positioned at inside (Armstrong etc. 1984, Rottier etc., 1986) thereby this albumen crosses over that viromembrane makes the outside that the N-end is positioned at virosome for three times.M has about 100 amino acid whose long cytoplasmic tails that may be embedded in the film.The M albumen of most of coronavirus is that N-or O-are glycosylated.

It is essential (Holmes etc., 1981, Rottier etc., 1981) that described M albumen forms for virosome.For example, the interaction between M and S is important (Opstelten etc., 1994,1995) for coating grain insertion virosome.In addition, the interaction between M and the N may be essential (Sturman etc., 1980) for core being incorporated into the budding virosome.

Little membranin E

Up to phase early 1990s, think that just the membranelle albumen E that is about 10kD is structural protein (Liu and Inglis, 1991).E is highly hydrophobic membranin but comprises many charged residues at the C-end.In TGEV, shown that this proteic C-end is positioned at the outside (Godet etc., 1992) of film.E as if both not by glycosylation also not by phosphorylation.Although be clear that E albumen is that its definite function in this process is waited until and illustrated for virosome assembling important protein (Vennema etc., 1996).

S protein

The spike protein of coronavirus is a 1100-1450 amino acid whose I type glycoprotein.The S albumen of some coronavirus is become two subunits, S1 and S2 with proteoclastic mode cracking.Yet, this cracked effect waited until illustrates.The spike protein sequence of more different coronavirus shows that the S2 subunit is than S1 subunit more conservative (Cavanagh, 1995).Be expected at the signal sequence of protein N-end, expection protein is cleaved after the film transposition in ER.35 potential N-glycosylation sites of as many as are present in the ectodomain of spike protein, but do not detect tangible fusion rotein.Identified the anchor of striding film of the carboxyl terminal that approaches spike protein.

MHV A59 as example, has been described the ripe and transhipment of coronavirus spike protein.At first, the quilt that spike protein is synthesized 120kD is being translated glycosylated protein (Niemann and Klenk, 1981) altogether.In addition, some in will 42 cysteine residues in ectodomain form intrachain disulfide linkage (Luytyes etc., 1987).The S monomer is slow oligomerization (Venemma etc., 1990 in the ER that may relate to some seven residue tumor-necrosis factor glycoproteins zone; Delmas and Laude etc., 1990).Before oligomerization, the monomeric M protein interaction among some in the S protein and the ER, this is the primary essential condition of bonded (Opstelten etc., 1993,1994) that arrived virosome afterwards.After correct the folding, compartment in the middle of spike protein is moved to, they are by palmitoylation therein.Middle compartment is budding site (Tooze etc., 1987) just.Because M and E are sufficient to the coating assembling (Venemma etc., 1996) of virus independently, only have with M bonded S protein to be inserted in the virus of budding (Opstelten etc., 1994,1995).The virosome of assembling uses the vesicle of composition approach to arrive the surface (Tooze etc., 1987) of infected cells.When vesicle and film fusion, the content of vesicle is released.

S protein is host cell tropism's major decision.Except being responsible for the fusion between peplos and the cytolemma, the virus protein that S is discerned by virus receptor, for example, the hCD13 (Aminopeptidase N) in HCoV 229E situation.

Epidemiology and pathogeny

The crown virus of end user's respiratory tract and chitling road has been described below epidemiology and the pathogeny of coronavirus as an example.At the beginning of the sixties in last century,, first the human corona virus is described as the reason of acute respiratory disease in the U.S. and Britain.In nineteen sixty-five, Tyrell etc. are separated to the B814 of human corona virus strain from a boy's suffering from flu nose irrigating solution.After 1 year, in Chicago, bacterial strain 229E is adapted to growth (Hamre and Prochnow, 1966) in tissue culture.In 1967, Macintosh etc. separated a series of strains that only can duplicate in organ cultures.In these strains, HCV OC43 and HCV 229E are considered to two of the human corona virus prototype of colony independently on serology now.

In EPDML a series of clinical studyes, shown that all acute respiratory diseases above 20% cause (Cavallaro and Monto, 1970 by coronavirus to coronavirus; Macnaughton etc., 1983).With rhinovirus, adenovirus and paramyxovirus, they are most important reason of such disease.Usually the human corona virus infects the epithelial cell of respiratory tract.With infecting related clinical symptom is to have a headache, have a fever, cough and sneeze.But, reported that coronavirus also can cause having the more respiratory disease of serious symptoms (Matsumoto and Kawana, 1992).Main bang path carries out by the aerosol of respiratory secretions or by mechanical transfer.And, shown 1 class human corona virus's member also relevant (Zhang etc., 1994) with gastrointestinal tract disease.Subinfection takes place in whole life stage again, shows that frequent inoculating needle is useful to the vaccine of coronavirus infection.

TGEV and PEDV can cause serious problems in the meat production industry to the infection of pig.The immunology research of carrying out from 1987 at Switzerland disclose 50% animal with acute bowel problems for PEDV be have seropositive.And the percentage range of every group of seropositive animals is in 17-100% (Hofmann and Wyler, 1987).The virus that infects is mainly transmitted by the ight soil of infected animal.The pathogenic effects of these coronavirus is that the diarrhoea by the pig of young pig, raising and the pig that fleshing up causes to a great extent.The clinical sign of infecting is to vomit sometimes and dejected watery diarrhea formerly.Epithelial infection and destruction have caused the dehydration of infected animal.The infection of youngling can cause their death.The closely similar mechanism of causing a disease of PED and TGE causes identical immunology situation.At the protection of the virus infection immunity based on intestinal mucosa, it is limited to metainfective short-term in the time.The lactagogue immunity, but be not that circulating antibody has protectiveness for the piggy of not weaning.Can not obtain the vaccine that to protect pig to avoid infecting now.

Immunne response at coronavirus infection

Inducing of humoral immunoresponse(HI)

Be primarily aimed at S by the generation of MHV mice infected, but also at M, the antibody of E and N.Some directly at the external neutralization virus of the monoclonal antibody of S, form (Grosse etc. 1993) although escape the virus mutation strain of neutralizing antibody in tissue culture.Kolb etc. proved antibody in the milk of recombinant animal and the natural generation by obtaining the lactagogue immunity those protection is delivered to the next generation.In Mammals, passive immunization is provided by the neutralizing antibody that placenta and milk are delivered to filial generation by the quilt as immunoglobulin G and excretory immunoglobulin A.Produced the genetically modified mouse of carrying encoding antibody light chain and heavy chain, described antibody can in and the neuralward JHM strain of murine hepatitis virus (MHV-JHM).MHV-JHM causes acute encephalitis and acute and chronic demyelination in the responsive strain of mouse and rat.Be disclosed in linear dependence between antibody expression and the virus neutralization activity derived from the analyzed in vitro of the milk of different transgenic strains, illustrate that recombinant antibodies is MHV-JHM neutral major decision during mouse is suckled.

In the former experiment, genetically modified filial generation and control mice are attacked by the MHV-JHM of lethal dose.Young baby (litter) by female beastly (dam) lactation of non-transgenic dies from fatal encephalitis, is avoided attacking and adequately protect by the young baby (litter) of female beastly (dam) lactation of transgenosis, no matter whether they are genetically modified.These experimental results show that the single neutralizing antibody of expressing is enough to thoroughly protect the filial generation of not wean to avoid MHV-JHM-inductive encephalitis in the milk of transgenic mice.

Inducing of cellullar immunologic response

In the past, some reports about the importance of the cellullar immunologic response of removing coronavirus infection have been arranged.Recently, the infectious segmental bronchus virus of report (IBV) such as Seo infects and relative disease can change greatly by the lymphocytic passive transport of immune T.Particularly, will before with virus attack, be transferred to young chicken at the lymphocyte that infection was collected in back 10 days.As determined by respiratory disease and virus quantity, the lymphocytic transfer of homology T protection chicken avoids challenge infection, and does not observe protection in the lymphocytic chicken of having accepted from infected chicken not of MHC consistency.In the chicken of the lymphocytic cell of alphabeta that is accepted in enrichment, observe virus infection and disease and almost removed fully.On the contrary, the lymphocytic removal of gammadelta T only protects the potential of chicken to have minimal effects for them.The lymphocytic transfer of the CD8 (+) of the enrichment of adopting or CD4 (+) T shows that protection also is the major function of carrying the cell of CD8.It is important protecting chicken to avoid in the IBV infection that these presentation of results have the antigenic alphabeta T of CD8 (+) lymphocyte.

Comprehensive these data, thus two arms of stimulating immune system protection host avoids infecting and removes viral useful after infection.Yet, will be appreciated that useful result also can obtain by an only arm of stimulating immune system.Embodiment of the present invention specifically are intended to the method for one and/or two arm of stimulating immune system.

Present vaccine method in veterinary science

Subunit vaccine

Coding cause the infectious peritonitis of cat coronavirus the fusion spike protein gene with genome (Vennema etc., the 1990) reorganization of vaccinia virus.This recombinant vectors is induced furcella-protein specific in mouse, external neutralizing antibody.Yet, when with recombinant virus immunity kitten, only obtained the neutralizing antibody of low titre.Like this, do not observe protection.

In second report of Venemma etc., to estimating with the similar immunity that nucleocapsid (N) albumen carries out with FIPV film (M).The vaccine virus recombinant chou of cloning by expression gene is induced antibody in by the kitten of immunity.The immunity of carrying out with N protein recombinant chou does not obviously influence the consequence of attacking.But, and there is one to compare in 8 kittens of control group, there are three after attack, to survive in 8 kittens with the immunity of M albumen recombinant chou.Yet because sample is very few, these quantity do not have the significance on the statistics.

Attenuated virus

In 1989, Christianson etc. developed temperature sensitivity (ts) FIPV strain, and it duplicates at 31 ℃, and does not duplicate at 39 ℃.This strain produces this strain passed for 99 generations continuously in tissue culture and simultaneous UV-irradiation after.In 1991 this ts strain market is turned to the FIPV vaccine that trade(brand)name is Primucell by Pfizer.Described vaccine is transmitted also because described virus is temperature sensitivity by approach in the nose, and it is only faint duplicating in the upper respiratory tract of " cold ".The strain of this attenuation may be the most effective coronavirus vaccine; Yet have a large amount of reports to point out to compare with control group, described vaccine is not being reduced the incidence that FIPV infects in the group of inoculating.

The virus of deactivation

In 1984, Cavanagh etc. reported the IBV protein inoculation chicken with the saccharose gradient purifying, attack the bird of inoculation then with IBV.Although S-protein causes antibody to produce, it is invalid in the chicken that IBV protection/resistance is given to be inoculated, as by they for typical IBV respiratory tract infection susceptibility proved.

Rotavec Corona is the combined vaccine of the marketization of the bovine rota, bovine coronavirus and the intestinal bacteria F5 (K99) that comprise deactivation.According to label, this product is not used in preventing infection, but is used to induce virus to discharge.Because this product can not be used for preventing infection, its efficient as vaccine is not very high.

In nearest area research, Takamura etc. (2002) use from the extract of the bovine coronavirus of infected cells and carry out intramuscular inoculation for the Holstein milk cow.Not not surprisingly, vaccine be (i) safety and (ii) can induce antibody response.Yet protected data is not included in the research report.

In brief, it seems that as if the virus formulation of deactivation be not the promising approach of exploitation at effective preventive measures of coronavirus infection.The invention provides the more effective approach of inducing at the immunne response of SARS virus.

Severe acute respiratory syndrome (SARS)

Since the year ends 2002, reported the serious atypical pneumonia of the unknown cause of disease of hundreds of examples in the Guangdong Province of the People's Republic of China (PRC).During in February, 2003 and March, in Hong Kong, Vietnam and Canadian patient, detect similar case after, The World Health Organization (WHO) has issued global for being called as " severe acute respiratory syndrome " disease alarm (SARS).To 2003 4 the end of month, surpass in the world 25 countries to WHO report surpass 4300 SARS cases and 250 SARS-associated deaths.

The seriousness of SARS influence is variable.For the cultivation period of this disease normally 2-7 days.The feature that infects is normally had a fever, and is dried after several days, non-productive cough and short of breath.The death of dying from the respiratory insufficiency of carrying out property occur in about 3% in 10% the case almost (Poutanen etc., 2003; Lee etc., 2003; Tsang etc., 2003).When new coronavirus (SARS-CoV) has been separated in the laboratory in the U.S., Canada, Germany and Hong Kong from SARS patient, identify that the etiologic trial of SARS outburst was achieved success in the 3rd week in March, 2003.Do not resemble other human corona virus, breeding SARS-CoV is possible in African green monkey kidney cell strain system.The evidence that SARS-CoV infects is embodied among the SARS patient in worldwide.In breath sample, frequently detect SARS-CoV RNA and comprise antibody with the SARS-CoV reaction from the serum sample in SARS rehabilitation of patients stage.Have the intensive evidence illustrate this new virus on epidemiology with the outburst of SARS be related (Ksiazek etc., 2003; Peiris etc., 2003; Drosten etc., 2003).Sequence (Rota etc., 2003 of two strain isolateds have been reported recently; Marra etc., 2003).Systems analysis and sequence relatively shown SARS-CoV and any before the coronavirus of identifying all keep off.

Reduce the system and method for the influence of SARS infection

Embodiments more of the present invention relate to and will carry SARS-CoV M, and the carrier of E and S gene is applied to induce immune response.In a preferred embodiment, described carrier is induced two arms of human immune system, and replying of body fluid and cell part still only induces the carrier of an arm also to be useful and to be specifically related in some embodiments of the present invention.The immunogen preparation that the present invention describes is safe, because dangerous SARS-CoV has no chance and can produce from be used to form immunogenic gene.In some embodiments, described DNA is delivered to people's cell, and wherein SARS virus albumen is expressed.Originate from SARS M, the spontaneous formation VLPs of the virus protein of E and S gene, it is secreted from cell, is secreted in course of infection as virus.The outer expression of inducing antibody response, the immunity system that therefore effective preparation SARS virus infects of existing of antigenic born of the same parents.In one embodiment, the immunne response of the gene induced cell of immunogenic SARS-CoV.

Plasmid vector

A kind of carrier that can be used for producing SARS-CoV-VLPs is a plasmid DNA.A large amount of plasmids are suitable for producing immunogen such as SARS-CoV-VLPs.Usually, be used to produce immunogenic plasmid and have the cloning site that is used to produce antigenic DNA insertion, the dna sequence dna that plasmid replication needs, the marker gene in that host cell is selected in such as bacterial cell promotes antigen expression promoter/enhanser and polyadenylation signal in eukaryotic cell.A large amount of this plasmids are commercially available and most of some or all of common features that have.In order to make the minimizing possibility of chromosomal integration, the homology of the sequence in plasmid DNA and the people's gene group is preferably limited.Antigenic expression can be driven by any suitable promotor or promotor/enhanser combination.For example, in some embodiments, express by the promotor/enhanser of the immediate early gene of cytomegalovirus (CMV) or from the promotor of Rous sarcoma virus (RSV) long terminal repeat (LTR) being driven.In some embodiments, kalamycin resistance gene can be used to select to have the intestinal bacteria of plasmid DNA separately, but but can use any suitable selective marker.Do not recommend to use beta-lactam antibiotics, for example, penbritin is because there is allergic report in some individualities.In some carriers, duplicating by pMBl (ColE1) replication orgin of the plasmid DNA in host bacterium regulated; Yet, can use any suitable underlying carrier starting point.

In some embodiments of the present invention, can (Invitrogen, Carlsbad CA) be used to express SARS-CoV M, E and/or S polypeptide with being used to develop the pVAX1 of dna vaccination by specific design.Each regulation of using (FDA, 1996) unanimity of the structure of pVAX1 and food and medicine.In addition, the small size of this carrier makes it be suitable for the antigenic cDNAs of a plurality of generations in a construct is carried out subclone.

Will be appreciated that the present invention is not limited to pVAX1.Can use other carrier, plasmid and non-plasmid.Other typical plasmid vector comprises, but be not limited to, Rapid VACC and pDNA-VACC (Nature Technology Corporation, Lincoln, NE) and other carrier for expression of eukaryon such as pSVL and pKSV-10 (Pharmacia), and pBPV-1/pML2d (InternationalBiotechnologies, Inc.), and pTDT1 (ATCC, #31255).

Virus vector

Typically, the attenuation RNA viruses of Huoing is highly effective for the purpose that excites immunogenic response.The attenuation rna virus vector of extremely successful work includes, but are not limited to the Sabin poliovirus, the 17D strain of Schwarz Measles virus (MV) and yellow fever virus.These viruses have been caused significantly reducing of corresponding infection and their related diseases Neo-Confucianism as vaccine.

For vaccinated purpose, the RNA viruses of attenuation has long security and effective record.In addition, these carriers be easy to produce, inexpensive and enjoy the system of distribution of broad range.When being used to produce immunogenic replying, after single low dosage injection, the Measles virus of attenuation is induced strong, body fluid and cellular immunity that the life-span is long.The MV genome is highly stable and never observe virus and reverse the cause of disease state.MV duplicates in tenuigenin exclusively, and therefore its genome never is incorporated on the host DNA.In addition, set up corresponding to the Edmonston of MV and the genomic infectious CDNA clones of Schwarz/Moraten strain.Set up the program of saving corresponding virus (EMBO J.145773-5784,1995).Will be in the cDNA of as many as 5kb on length successful expression in these carriers.Therefore, the attenuation of living, reorganization MV virus vector is to be used for exciting the two the potential good carrier at measles and SARS the crowd.

Alternative as plasmid DNA can be transmitted one or more purposes SARS-CoV gene as carrier with recombinant virus.For example, in some embodiments of the present invention, use recombinant measles virus.Can be used to produce recombinant measles virus as the cDNAs that this paper describes the clone who is prepared.In some embodiments of the present invention, recombinant virus only carries one of them of SARS-CoV cDNAs.In other embodiment, recombinant virus carries coding M, two or more cDNAs of S or E polypeptide.

In some embodiments of the present invention, will be corresponding to SARS-CoV M, the cDNAs of one or more of E or S gene is cloned in the Measles virus carrier, and it represents the recombinant cDNA plasmid form of the geneome RNA of Measles virus.Recombinant virus can produce from these plasmids by the rescue experiment of the standard in tissue culture (Takeda etc., 2000).

Will be appreciated that the embodiment based on virus of the present invention is not limited to the application of MV carrier.Other exemplary virus vector includes, but not limited to retrovirus, adenovirus, adeno associated virus, and lentiviral vectors.

Prokaryotic vector

The attenuated bacteria of living is allowed and is a kind ofly carried out the antigen transmission and immunogenicity stimulates and selectively targeted alternative approach to the antigen presenting cell on immune inducibility site by mucous membrane surface.A kind of method utilization is with the intracellular bacteria of the attenuation transfer system as eucaryon antigen presentation carrier.Candidate's carrier bacterium includes, but not limited to the attenuated strain that salmonella (Salmonella), Shigella (Shigella) and listeria spp belong to (Listeria) kind.The former demonstration of some member in these kinds immunogenic antigenic DNA that will encode passes to people's cell.It is effective that the transmission of the DNA of coding for antigens and the generation of immunogenic response have proved in the experimental animal model of several transmissible diseases and tumour.

For effectively, the protokaryon bacterial strain of the attenuation of living be kept balance between attenuation and immunogenicity.Thereby these bacterial strains do not cause any disease or weaken host's physiology and can settle down after Orally administered in the intestines Lymphoid tissue relevant with digestive tube at the same time or settle down in other lymphoid organ to have immunogenicity after using by some other approach.As antigen vectors, it is useful especiallyly (, to see Essentials ofMusocal Immunology such as Curtiss for summary that recombinant salmonella has been presented in the living vaccine, Kagnoff and Kiyono, Eds., Academic Press, San Diego, 1996, pp.599-611; Doggett and Brown, Mucosal Vaccines, Kiyono etc., Eds., Academic Press, San Diego, 1996pp 105-118; Also see Infect Immun.63:3279-3286 such as Hopkins, 1995; Vaccines such as Srinavasin 95, R.N.Chanock etc., Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y., p 273-280,1995).When being used as recombinant antigen and transmitting media, with the attenuated strain of salmonella typhi (Salmonella typhi) as at typhoid fever and at people's vaccine of heterologous antigen.(Forrest, CRC PressInc., 1994, pp.59-80; Levine etc., New Generation Vaccines Woodrow andLevine, Eds., Marcel Decker, Inc., New York, 1990, pp.269-287).

In some embodiments of the present invention, the salmonella typhi with attenuation is used for the required gene of encoding SARS-CoV antigen protein is passed to the people.Described method comprises the bacterial strain of selecting bacteria such as salmonella typhi, described bacterial strain has short natural death of cerebral cells (pro-apoptotic) gene of (i) deactivation, (ii) the vacuole of deactivation is kept one or more the deactivation sudden change that gene (iii) makes the bacterial strain attenuation, (iv) encoding SARS-CoV S-, the recombination of the polypeptide of M-and E-.Then described bacterial strain is administered to the people.Make the mutant of one or more deactivations of bacterial strain attenuation can in a gene, comprise sudden change or in each of two or more genes, comprise sudden change.In addition, the salmonella strain of attenuation comprises at least one recombination that can express the SARS-CoV gene, and it is allowed them as gene product is passed to the experimenter, such as people's carrier or transmission media.As for transmitting required gene, mean nucleic acid with encoding SARS-CoV product, DNA or RNA pass to the experimenter.The salmonella bacterial strain also can transmit corresponding to virus replication or infectious, the virus of attenuation such as, but be not limited to Sabin poliovirus, the RNA of yellow fever virus 17D or Measles virus.When transmitting in the tenuigenin of viral RNA in infected cells, virus duplicate or replicon begins and as the result that duplicates, antigen sequence is further increased and observes better expression.

In some embodiments of the present invention, the application of salmonella typhi promotes to be collectively referred to as the gut-associated lymphoid tissue (GALT) of mucous membrane associated lymphoid tissue, the invasion of any of nose associated lymphoid tissue (NALT) or bronchus associated lymphoid tissue (BALT) and settling down.In some advantages of using salmonella typhi to obtain in as transfer system, if be that bacterium can make required SARS-CoV gene product by Orally administered transmission or be settled in gut associated lymphatic tissue, if if by the intranasal administration transmission or be settled in the nose Lymphoid tissue and use the fact of transmitting or being settled in other lymphoid organ by other approach.In addition, the salmonella typhi of application attenuation is a kind of for the effective and inexpensive method that nucleic acid molecule is delivered to people's cell.

In some embodiments of the present invention, the Salmonellas of attenuation can be settled in aggregate lymphatic nodule or comprise, the adenoid similar tissue of other GALT in the people for example, and can not destroy invaded cell.This act on Orally administered after, high immunogenicity is provided.And, the adenoid M cell of company's folliculus of GALT is on function, on the form and structure on and with intravital other mucous membrane dependency lymphoid tissue (MALT), such as conjunctiva associated lymphoid tissue (CALT), bronchus associated lymphoid tissue (BALT) and nose associated lymphoid tissue (NALT), and relevant M cell such as the Lymphoid tissue in rectum is identical.So, when using by suitable way, for example mouthful, in the nose and during rectum, these all tissues can be invaded and be settled in to Salmonellas.

At inducing of the immunne response of SARS-CoV-VLPs

In some embodiments of the present invention, in the experimenter of risky development SARS, induce immunne response at SARS-CoV-VLPs.These experimenters comprise animal such as bird and Mammals.Embodiments more of the present invention relate to the immunne response of inducing at SARS-CoV-VLPs in chicken and other poultry.In some embodiments, induce the immunne response at SARS-CoV-VLPs in Mammals, described Mammals includes, but not limited to mouse, rat, cat, dog, pig, ox, horse, goat, sheep and monkey.In a preferred embodiment, in the people, induce immunne response at SARS-CoV-VLPs.

Be intended to can be in fact the carrier of any kind that comprises the naked DNA of plasmid or other nucleic acid carrier form is used for uniting the generation immunne response with the immunization protocol of broad variety, described immunization protocol includes, but not limited to parenteral, mucous membrane and gene gun inoculation.

Immunogenic preparation and coding are normally better understood this area as the immunogenic genetic constructs of activeconstituents, as by U.S. Patent number 6,716,823; 5,703,057; 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; With 4,578, institute's illustration in 770.Typically, with SARS-CoV-VLPs, the prokaryotic vector that coding can form the genetic constructs of the SARS-CoV polypeptide of SARS-CoV-VLPs or its part and/or comprise this genetic constructs is prepared into injectable, liquor or suspension, or be mixed with the solid form that becomes solution or suspension before being adapted at injecting.Also these preparations can be carried out emulsification.The composition of active immne originality is often mixed with medicinal and compatible with activeconstituents vehicle.Suitable vehicle is for example water, salt solution, glucose, glycerine, ethanol or analogue and combination thereof.In addition, if desired, described preparation can comprise auxiliary substance such as wetting agent or emulsifying agent, pH buffer reagent in a small amount, or increases the adjuvant of the immunogen usefulness of preparation.

The immunogen preparation routine can be passed through, for example, inject hypodermically or intramuscularly, use in the stomach other places.The alternate manner that is suitable for using comprises the powder of intranasal administration, oral preparations and suppository.

Usually when having the vehicle of excess powderization, by in the aqueous solution of hydrophobic vehicle, suspending insoluble nucleic acid construct or VLPs and described solution drying comprised the nucleic acid construct that is dispersed in the exsiccant excipient materials or the powder of VLPs with generation, prepare the powder that nose is used.In starting soln, nucleic acid construct or VLP are any ratios consistent with the application that is intended to the weight ratio of hydrophilic excipient.Preferably, in starting soln, the ratio of nucleic acid construct or VLP and hydrophilic excipient is 1: 1-1: 10.Can subsequently it be ground to produce whole powder by with droplet jet (spraying drying) or carry out drying to produce coarse meal in airflow flowing by vacuum-drying.Be used for the particulate situation that lung transmits in intention, the particle with 0.5 μ m-5 μ m size is an ideal.

Oral preparations comprises these normally used vehicle, as, for example, the mannitol of pharmaceutically grade, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.These compositions can take solution, suspension, tablet, pill, capsule, sustained release dosage or powder and, in some embodiments, comprise the active ingredient of about 10-95%, preferably about 25-about 70%.

For suppository, traditional tackiness agent and carrier can comprise, for example, and polyalkalene glycol or triglyceride level.These suppositorys can form the mixture of self-contained activeconstituents.In some embodiments, it is about 10% at about 0.5%-that described suppository forms self-contained scope, the mixture of the active ingredient of preferably about 1-about 2%.

In some embodiments of the present invention, in the mode compatible and to be immunogenic and in treatment, effectively to measure and use immunogenic preparation with formulation.The amount of using depends on experimenter to be treated, comprises, for example, the ability of experimenter's immuning system synthesising antibody and required degree of protection.The accurate amount of the activeconstituents that need be applied depends on professional's judgement.Yet in preferred embodiments, suitable dosage ranges is the grade of hundreds of micrograms of every dosage.The suitable scheme of initial administration and booster dose also are variable, but by the initial representative of using, carry out subsequently afterwards inoculation or other use.

In many situations, multiple the using with immunological reagent will be ideal.In some embodiments, use and will normally be interval with 2-12 week, but more generally with the interval in 3-5 week.With 1-5, normally the reinforcement of the cycle in 3 years is an ideal for the protection level of keeping immunogenic response.Can mensuration after the immunologic process for the antibody of target antigen.Described mensuration can pass through to use conventional mark, such as, radionuclide, enzyme, fluorescence etc. carry out mark.These technology are well-known and are found in the patent of broad variety, such as U.S. Patent number 3,791,932; 4,174,384 and 3,949,064.

Embodiment

Embodiments more of the present invention further illustrate by not being considered to restrictive following embodiment.Skilled person will appreciate that in the practice of the embodiment of disclosed technology representative in invention described herein in embodiment subsequently to have the technology of good function, and therefore can think that it forms the preferred mode of the enforcement of these embodiments.Yet about this specification sheets, those skilled in the art will be appreciated that and can make many variations and still obtain similar or similar result in specific embodiments under the situation that does not deviate from spirit and scope of the invention.

Embodiment 1

Compare SARS-CoV M, E and S polypeptide from four SARS strain isolateds

Carry out the M for known SARS-CoV cause of disease strain, the analysis of the relation between E and the S polypeptide effectively is used in the potentiality of generation at the SARS-CoV-VLP that can extensively adapt to of the immunogenic response of most of or all SARS-CoV strain isolateds to determine exploitation.

The SARS-CoV strain isolated of use in this analysis is Tor2, Urbani, HKU-39849 and CUHK-W1.At the genome scientific center, British Columbia DKFZ, 600West 10thAvenue, Vancouver, BC V5Z 4E6, Canada separates SARS-CoV strain Tor2.Determine described genomic sequence, then with registration number: AY274119 (complete genome group, 29751bp) (SEQ ID NO:14)) is kept at it among Genbank.SARS-CoV strain Urbani is located away from Division of Viral and Rickettsial Diseases, Centers for Disease Control andPrevention, and 1600Clifton RD, NE, Atlanta, GA 30333, USA.Determine described genome sequence, (the complete genome group, 29727bp) (SEQID NO:15) is kept among the Genbank with registration number: AY278741 with it then.SARS-CoV strain HKU-39849 is located away from animal system, Hong Kong University, and Pokfulam Road, Hong Kong, HK 00000, China.Determine described genome sequence, then, (the complete genome group, 29742bp) (SEQ ID NO:16) is kept among the Genbank with registration number: AY278491 with it.SARS-CoV strain CUHK-W1 is located away from department of biochemistry, Hong Kong Chinese University, MMW Bldg.Rm 608, Shatin, NT SAR, China.Determine genomic sequence, then with registration number: (the complete genome group, 29736bp) (SEQ ID NO:17) is kept among the Genbank AY278554.

The other strain that has separated SARS-CoV, still the genome to them has only carried out partly order-checking.These strains are not included in this analysis, but are described below: SARS-CoV strain BJ01, BJ02, BJ03, BJ04 separate from microorganism and epidemiological study institute, Chinese Military Medical Science Institute/Beijing Joint Genome Institute, the Chinese Academy of Sciences, Beijing 101300, China.Each genome of these strains is carried out the part order-checking and respectively with registration number: AY278488, AY278487, AY278490, AY279354 is kept among the Genbank.SARS-CoV strain GZ01 separates from microorganism and epidemiological study institute, Chinese Military Medical Science Institute/Beijing Joint Genome Institute, the Chinese Academy of Sciences, Beijing 101300, China.The genome of this strain is carried out the part order-checking and with registration number: AY278489 is kept among the Genbank.

Relatively determine M, E and S amino acid sequence of polypeptide by the ORF analysis with the homology of other known coronavirus from genome sequence.For each of four strains, the sequence alignment program AlignX that use has blosum62mt2matrix comes comparison M, the sequence of E and S polypeptide, its open point penalty (opening penalty) is 10, it is 0.05 that point penalty (gap extension penalty) is expanded in the room.E, the sequence alignment of M and S polypeptide is shown in Fig. 4 A-C respectively.

The result of sequence parallelism shows M, and E and S polypeptide are high conservatives in four SARS-CoV strains of this paper test.The E peptide sequence show that relatively sequence is 100% identical (Fig. 4 A) for each of four SARS-CoV strain isolateds.Described M peptide sequence only shows small variation in four strains.Particularly, the strain HKU-39849 place that is different from other strain is on 67 positions it is Xie Ansuan rather than L-Ala.The place that strain Urbani is different from other strain is that position 154 comprises proline(Pro) rather than Serine (seeing Fig. 4 B).Described S polypeptide has also only shown subtle change.Particularly, strain CUHK-W1 77 comprises aspartic acid rather than glycine in the position, comprises Threonine rather than Isoleucine on position 244.Strain Tor2 577 comprises L-Ala rather than Serine (seeing Fig. 4 C) in the position.

Sequence presentation of results relatively forms from any the M from the strain of this paper test, and the VLP of E and S polypeptide will produce specificity not only and make up from wherein strain also at the immunne response of a large amount of different SARS-CoV strains at VLP.

Although embodiments more of the present invention relate to the gene constructed VLPs of use from single SARS-CoV strain, will be appreciated that other embodiment is intended to the gene from a plurality of SARS-CoV strains is applied to produce VLPs.In addition, other embodiment of the present invention relates to the mixture that comprises corresponding to the VLPs of a plurality of SARS-CoV strains, wherein produces each VLP by the gene that uses single SARS-CoV strain.

Embodiment 2

Preparation SARS-CoV cDNA

In this embodiment and the following examples, with the reverse RNA prepared product of transcribing of SARS-CoV strain Urbani cDNAs from SARS-CoV strain Urbani geneome RNA.Reverse method of transcribing is well-known in the art.The cDNA of preparation is used as the template of PCR reaction and other application described herein.Table 2 has been listed the Sequence Identification number (SEQ IDNO) of the cDNAs that is used for this paper.

Table 2: be used in the cDNAs in the plasmid construction

cDNA	SEQ ID NO：
		The proteinic cDNA of S-	SEQ ID NO：18
The proteinic cDNA of M-	SEQ ID NO：19
		The proteinic cDNA of E-	SEQ ID NO：20
The cDNA of IRES	SEQ ID NO：21
		The cDNA of RSV-LTR promotor	SEQ ID NO：22
The cDNA of SV40 polyadenylation signal	SEQ ID NO：23

Embodiment 3

SARS-CoV M, the plasmid of E and S polypeptide are expressed in preparation

In this embodiment, made up the single plasmid of expressing three all SARS-CoV polypeptide.

Use following primer:

RSVBACK：5’-AATAACTGCAGCGATGTACGGGCCAGATATAC-3’(SEQ ID NO：24)；

RSVFOR：5’-AATAAGCGGCCGCGGAGGTGCACACCAATGTGG-3’(SEQ ID NO：25).

(SEQ ID NO:25) comprises 411 bp dna fragmentations of Rous sarcoma virus (RSV) long terminal repeat (LTR) promotor by polymerase chain reaction (PCR) (Sambrook etc., 2001) amplification.

Thereby 5 '-end of the PCR product that the design primer will appear at the PstI-site to be obtained, and the NotI-site will appear at 3 '-end.These restriction sites allow that the RSV-LTR promotor is inserted in pVAX1 (Invitrogen, Carlsbad, CA) the unique Pst-and the NotI-site of the multiple clone site of (SEQ ID NO:26).

Except the RSV-LTR promotor, use following primer:

SV40BACK：5’-TTATTAAGCTTATGTACTCATICGTTTCGGAAG-3’(SEQ ID NO：27)；

SV40FOR：5’-TATTGGTACCGACCAGAAGATCAGGAACTCC-3’

(SEQ ID NO:28) comprises the 240bp dna fragmentation of simian virus (SV) 40 polyadenylation signals by pcr amplification.

Use the amplification of above-mentioned primer the HindIII-site is added to the SV40 that obtains polyadenylation signal 5 '-terminal and the KpnI-site is added to 3 '-end.Then, this PCR fragment is inserted the unique HindIII and the KpnI-site of the pVAX1-derivative that has comprised the RSV-LTR promotor.The plasmid that obtains is called pCSRB.

Use following primer:

SARS-S-BACK：

5’-TAATATCTAGAGCCGCCGCCATGTTTATTTTCTTATTATTTCTTACTCTCAC-3’(SEQID NO：29)；and

SARS-S-FOR：

5’-TAATAGTTTAAACTTATCATGTGTAATGTAATTTGACACCC3’(SEQ ID NO：30).

By pcr amplification encoding SARS-Cov strain Urbani (Genbank registration number: the cDNA fragment (SARS_S) of the 3792bp of furcella AY278741) (S) glycoprotein.

Use above-mentioned primer, with 5 '-end of XbaI-site and Kozak (Kozak, 1987) consensus sequence adding SARS_S cDNA PCR product.Described Kozak sequence is the recognition sequence of the weak point found in most of Eukaryotic mRNAs, and it promotes combining of initial mRNA and small subunit ribosome greatly.The consensus sequence of initial translation initiation is (GCC) GCC in the vertebrates ^A/ _GCCATGG (SEQ ID NO:31).At 3 '-end of SARS_S cDNA, added two translation stop codon and PmeI-site and made this sequence be inserted in the unique XbaI-and the PmeI-site in the RSV-LTR promotor downstream among the pCSRB as the Xbal/PmeI-fragment thus.

Finally, use following primer:

SARS-M-BACK：

5’-TAATAGCTAGCGCCGCCGCCATGGCAGACAACGGTACTATTAC-3’(SEQ ID NO：32)；

SARS-M-FOR1：

5’-GAGCTGTTTTAATCATTACTGTACTAGCAAAGCAATATTGTC-3’(SEQ ID NO：33)；

IRES-BACK：5’-GCTAGTACAGTTAAAACAGCTCTGGGGTTGTAC-3’(SEQ ID NO：34)；

IRES-FOR：5’-CGAATGAGTACATTATGATACAATTGTCTGATTG-3’(SEQ ID NO：35)；

SARS-E-BACK1：5’-TTGTATCATAATGTACTCATTCGTTTCGGAAG-3’(SEQ ID NO：36)；and

SARS-E-FOR1：

5’-TATTACTTAAGTTATCAGACCAGAAGATCAGGAACTCC-3’(SEQ ID NO：37).

By PCR the cDNAS (SARS_E cDNA) of the cDNAS (SARS_M cDNA) of encoding SARS-CoV M polypeptide and coding E-polypeptide and the fragment that comprises Mahoney strain poliovirus type I internal ribosome entry site (IRES) are increased.

Thereby the design primer is overlapping introducing～25bp's between 5 '-end of 3 ' of SARS_M cDNA-end and IRES and between 5 '-end of 3 ' of IRES-end and SARS_E CDNA.In addition, the 5 '-end that NheI-site and Kozak consensus sequence is added SARS_M cDNA product.Two translation stop codon are added 3 '-end of SARS_M cDNA product and 3 '-end of two translation stop codons are sub and AflII-site introducing SARS_E cDNA product.In final PCR reaction, these three fragments are connected by overlapping extension, cause being called the fragment of M_IRES_E by this paper.Use restriction enzyme NheI and AflII, M_IRES_E is positioned at the downstream of the CMV promotor of the pCSRB-derivative that comprises SARS-S cDNA.The construct that obtains is called pMES.

Embodiment 4

The plasmid of SARS-CoV M and E polypeptide is expressed in preparation

In this embodiment, use the plasmid of pVAX1 construction expression SARS-CoV M and E polypeptide.With the plasmid called after pME that obtains.Make up pME by encoding from the M of SARS-CoV and the polylinker zone of the proteinic bicistronic mRNA construct insertion of E pVAX1.Use following primer:

SARS-M-BACK：

5’-TAATAGCTAGCGCCGCCGCCATGGCAGACAACGGTACTATTAC-3’(SEQ ID NO：38)；

and

SARS-E-FOR2：

5’-TATTAGTTTAAACTTATCAGACCAGAAGATCAGGAACTCC-3’(SEQ ID NO：39).

Prepare M/E bicistronic mRNA construct by the M_IRES_E fragment that increases again from pMES (seeing embodiment 3).

The amplification of carrying out with SARS-M-BACK and SARS-E-FOR2 produces the M_IRES_E fragment, and it is added the NheI-site and comprise the PmeI-site at its 3 '-end its 5 '-terminal transformation.The unique NheI-and the PmeI-site of 3 '-end of multiple clone site that described fragment is inserted in the pVAX1 in CMV promotor downstream allowed in these sites, produces pME thus.

Embodiment 5

The plasmid of SARS-CoV S polypeptide is expressed in preparation

In this embodiment, use the plasmid of pVAX1 construction expression SARS-CoV S polypeptide.With this construct called after pS.By will as the segmental SARS_S cDNA of XbaI/PmeI-(such as embodiment 3 generation) NheI-and the PmeI-site of inserting the CMV promotor downstream among the pVAX1 make up pS.Notice that XbaI and NheI produce match outstanding.

Embodiment 6

The plasmid of SARS-CoV M polypeptide is expressed in preparation

In this embodiment, use the plasmid of pVAX1 construction expression SARS-CoV M polypeptide.With this construct called after pM.In order to produce pM, use following primer:

SARS-M-BACK：

5’-TAATAGCTAGCGCCGCCGCCATGGCAGACAACGGTACTATTAC-3’(SEQ ID NO：40)；

SARS-M-FOR2：

5’-TAATAGTTTAAACTCATTACTGTACTAGCAAAGCAATATTGTC-3’(SEQ ID NO：41).

By pcr amplification SARS_M cDNA.

Then, SARS_M cDNA product is connected to the multiple clone site of the pVAX1 in CMV promotor downstream as the NheI/PmeI-fragment, produces plasmid thus, pM.

Embodiment 6

The plasmid of SARS-CoV E polypeptide is expressed in preparation

In this embodiment, use the plasmid of pVAX1 construction expression SARS-CoV E polypeptide.With this construct called after pE.In order to produce pE, use following primer:

SARS-E-BACK2：

5’-TTATTGCTAGCATGTACTCATTCGTTTCGGAAG-3’(SEQ ID NO：42)；and

SARS-E-FOR2：

5’-TATTAGTTTAAACTTATCAGACCAGAAGATCAGGAACTCC-3’(SEQ ID NO：43).

By pcr amplification SARS_E cDNA.

Then SARS_E cDNA product is connected to the multiple clone site of the pVAX 1 in CMV promotor downstream as the NheI/PmeI-fragment, produces plasmid thus, pE.

Embodiment 7

The virus of SARS-CoV polypeptide is expressed in preparation

This embodiment describes the structure of the recombinant measles virus (MV) of expressing the SARS-CoV gene.

The MV recombinant plasmid is derived from plasmid p (+) MV, and it carries the Schwarz/Moraten vaccine strain of anti-genomic MV mark Edmonston B or MV sequence.Two other transcription units (ATU) that will comprise the restriction enzyme site that inserts open reading-frame (ORF) (ORFs) introduce MVcDNAs.First ATU is positioned at the downstream of P gene and the downstream that second ATU is positioned at the H gene.The SARS-CoV gene that the plasmid of these through engineering approaches is used to insert coding M and E polypeptide.

Use the Pfu polysaccharase and comprise and come amplification coding S protein and the proteinic SARS-CoV cDNAs of M/E (seeing embodiment 3) by PCR for being cloned into the unique BsiWI in the MV carrier and the primer in BssHII site subsequently.The described primer artificial initial sum terminator codon of also encoding.Comprise after the terminator codon that thereby other Nucleotide meets " 6 rule (rule of six) ", described " 6 rules " require the genomic Nucleotide number of MV must be 6 multiple.SARS CoV structure gene is introduced in first and second the ATU site of pMV carrier.

Describe (EMBO J.145773-5784,1995) by Radecke etc., use rescue system from plasmid, to reclaim the MV-SARS-CoV virus of recombinating based on helper.In brief, use the calcium phosphate method, with plasmid co-transfection stably express t7 rna polymerase and the measles N and the proteinic people's helper of P of different pMV/SARS-CoV gene plasmid (5 μ g) and expression MV polysaccharase L gene.Behind 37 ℃ of incubations that spend the night, carry out heat-shocked (43 ℃ 2 hours) with the substratum of fresh substratum displacement transfection and with cell.In 37 ℃ of incubations two days later, the African green monkey kidney cell strain that converges with transfected cell transfer to 70% is a cellular layer and in 37 ℃ of incubations.Cultivate after 2-5 days, it is in the cell that synplasm (syncytia) appears at the African green monkey kidney cell strain.The African green monkey kidney cell strain of single synplasm being transferred to the 35-mm diameter is in the hole of cell.Infected cells is expanded in T-75 or the T-150 flask then.When synplasm reaches 80-90%, collect virus by scraping cell in the MEM of 3ml substratum, carry out taking turns freeze thawing subsequently.From cell debris, clarified and be stored in-80 ℃ by the centrifugal supernatant liquor that makes then.

Embodiment 8

The prokaryotic vector of construction expression SARS-CoV polypeptide

In this embodiment, the salmonella typhi with attenuation is used to transmit SARS-CoV M, E and S gene.At pS, pM is used to produce with clone cDNAs among the pE and carries the special replication orgin of different Salmonellass (ori) but and selective marker, for example plasmid of kantlex with above-mentioned.Then, in tissue culture, test the generation of recombinant bacteria for the SARS-CoV specific protein.Then, the Salmonella typhimurium bacteria strain of attenuation is used for the M with SARS-CoV, E and S gene pass to the people.

Embodiment 9

Antibody at the SARS-CoV polypeptide

This embodiment describes can be in conjunction with SARS-CoV M, the generation of the polyclonal antibody of E and/or S polypeptide.Particularly, ripe SARS-CoV M, 15 amino acid of the aminoterminal of each of E and S polypeptide and carboxyl terminal chemically synthesize and are used for subsequently immunize rabbit.Used sequence is as follows:

S protein N-terminal: DLDRCTTFDDVQAPN (SEQ ID NO:44)

S protein/C-end: DDSEPVLKGVKLHYT (SEQ ID NO:45)

M protein N-terminal: MADNGTITVEELKQL (SEQ ID NO:46)

M protein/C-end: TDHAGSNDNIALLVQ (SEQ ID NO:47)

E protein N-terminal: MYSFVSEETGTLIVN (SEQ ID NO:48)

E protein/C-end: VKNLNSSEGVPDLLV (SEQ ID NO:49)

Peptide with 200 μ g excites by the animal of immunity at every turn, and 1 time every month, 3 times altogether.In experiment subsequently, the serum that obtains is used as M, the instrument of E and S polypeptide of detecting.

Embodiment 10

Express the SARS-CoV polypeptide

In each clone and the expression of SARS-CoV polypeptide of external check from construct described herein.For the structure of plasmid in pVAX1, the phage t7 promotor promotes the mRNAs by the external composite coding SARS-CoV of t7 rna polymerase antigenic peptide.At the antigenic mRNAs of in-vitro transcription encoding SARS-CoV, exist used as working as then ³⁵The S-methionine(Met) and/or ³⁵During the S-halfcystine, the template of the external translation reaction in reticulocyte lysate.Polyacrylamide gel electrophoresis (PAGE) by standard separates the SARS-CoV antigen of radiolabeled translation, and manifests by fluorography.Perhaps, use at M, E, and/or the antiserum(antisera) that the S polypeptide produces makes radiolabeled antigen carry out immunoprecipitation (seeing embodiment 9).By a-protein or protein G agarose precipitation immunocomplex, separate and manifest by fluorography by PAGE.

Can in tissue culture, check the functional of plasmid construction body described herein.In these experiments, any combination that produces the genetic constructs of the necessary polypeptide of SARS-CoV-VLPs with coding comes the transfection tissue culture cells.For example, available pMES transfectional cell; With pME and pS it is carried out cotransfection; Use pM, pE and pS carry out cotransfection.In these experiments, use fat transfection amine (Invitrogen) to come transfection about 2 * 10 according to manufacturer's handbook ⁶Cell.After 24 hours, lysing cell in containing the polyacrylamide gel sample buffer of SDS.Then, protein is separated in the 8-20%PAA gel that is purchased and through western blotting.After transferring on the soluble cotton, the rabbit anti-serum that will describe in embodiment 9 is used for detecting the SARS-CoV polypeptide of expressing at tissue culture cells.

After Fig. 5 was presented at the transfection of plasmid, transfected tissue culture cells produced SARS-CoV M, and E and S polypeptide, described plasmid are carried at these SARS-CoV genes under the eukaryotic promoter control.

The virus formulation body that comprises the SARS-CoV polypeptide can be used for cells infected culture and as these polypeptide expression of above-mentioned check.Fig. 6 illustrates after usefulness is carried the recombinant measles virus transfection of these SARS-CoV genes, and the tissue culture cell produces SARS-CoV M, E and S polypeptide.

Also can be in the histocyte culture, the protokaryon that check comprises genetic constructs transmits the vector construction body, such as the ability of inducing the SARS-CoV protein expression of plasmid as herein described.Fig. 7 for example understands tissue culture cells, and described cell is producing SARS-CoV protein with the attenuation salmonella bacterial strain transduction back of carrying genetic constructs, and described construct has SARS-CoV M, E and the S gene under eukaryotic promoter control.

Embodiment 11

Preparation SARS-CoV-VLP

Present embodiment proof M, E and S protein are enough to produce SARS-CoV sample particle, and it comprises three all protein by emiocytosis and described VLP.Described in embodiment in front, with plasmid DNA transfection or with recombinant virus infection or with carrying SARS-CoV M, the recombinant bacteria of E and S gene transduces about 2 * 10 ⁷The tissue culture cell.With about 24 hours of cell culture incubation, collect supernatant liquor then.In 24 hours cultivation stages, the SARS-CoV polypeptide is produced by cell, and virus-like particle forms, and budding takes place and VLPs discharges by cell.Fig. 8 illustrates the tissue culture cell, and described cell produces SARS-CoV-VLPs expressing SARS-CoV M behind E and the S gene, then these VLPs is discharged in the tissue culture supernatant liquor.

For M is described, E and S polypeptide all are the parts of VLPs, have used the density centrifugation that is untreated with dissolved cell conditioned medium liquid prepared product.For the dissolved prepared product, the aliquots containig that Triton-X 100 is added supernatant liquor is 1% to final concentration, therefore effective lytic virus coating.After the dissolving, M, E and S polypeptide freely pass through gradient according to their molecular weight and move.In contrast, the protein in untreated material is according to the more high molecular migration of VLP.Fig. 9 illustrates the density gradient centrifugation and the migration of dissolved protein (Triton-X100) in saccharose gradient of the complete virion of Triton-X100 of no use (nothing) processing.

Collection is analyzed corresponding to the fraction in the single zone of gradient and by aforesaid western blotting.This result shows: (i) SARS-CoV-VLPs is by being used the SARS-CoV gene M, and the eukaryotic cell of E and S transfection produces; (ii) all SARS-CoV polypeptide are the parts of SARS-CoV-VLP.

Will be appreciated that the method well-known in the art of using, SARS-CoV-VLPs can be separated from culture medium for tissue culture and it is mixed with immunogenic pharmaceutical preparation.Perhaps, all or part that produces the gene of the required coded polypeptide of SARS-CoV-VLP can be administered to the experimenter by method described herein.Expression of gene in the experimenter will be allowed the generation of immunogenic SARS-CoV-VLPs.

Embodiment 12

Pass to experimenter's carrier formulation

The preparation of plasmid vector

By using ethanol sedimentation and can preparing the plasmid DNA that is used to transmit by centrifugal collection plasmid.Use 70% ethanol that plasmid is thoroughly washed subsequently and simple dry.Any appropriate formulation can be used to use DNA.In one embodiment, DNA is dissolved then in phosphate buffer soln can use it for injection.

As alternatives, plasmid DNA can be mixed with the exsiccant powder.Exsiccant powder nucleic acid composition comprises that insoluble nucleic acid construct (typically small-particle) in the matrix that is dispersed in the hydrophilic excipient material is to form big aerosol particle.For the lung transmission, the aerosol particle of powdered has the mean particle size in 0.5 μ m-5 mu m range usually, and bigger size is effective for being delivered to other moistening target location.

The dry powder nucleic acid composition is prepared as follows: usually when having the vehicle of excess powderization, insoluble nucleic acid construct is suspended in the aqueous solution of hydrophilic excipient and dry described solution produces, is included in the particulate powder of dispersive nucleic acid construct in the exsiccant excipient materials.The nucleic acid construct in starting soln and the weight ratio of hydrophilic excipient are preferably from 1: 1-1: 10, and described solution can grind to produce whole powder it subsequently by liquid droplets to airflow flowing (spraying drying) or carry out drying by vacuum-drying and produce coarse meal.

At the particle of the granular size with 0.5 μ m-5 μ m of preparing to be used for the lung transmission, each particle comprises 10 usually ³-10 ⁴Nucleic acid construct.Described construct evenly or unevenly can be dispersed in each particle, and described particle successively will be usually with usually 1: 10-1: 500 weight ratio (nucleic acid construct: the excipient powders that is free on nucleic acid) in the excessive vehicle that is present in powdered.

The method of transmitting nucleic acid construct comprises that the dried powder that will comprise nucleic acid construct is oriented to the wet target location among the host, wherein when being exposed to wet target site, described particulate hydrophilic excipient substrate material will dissolve, and littler nucleic acid construct particle and cell are freely interacted.In a preferred embodiment, target location is that lung and described particle are oriented to lung by suction.

Dry powder compositions is particularly advantageous, because hydrophilic excipient will be stablized nucleic acid construct in order to storing.Excessive powdered hydrophilic excipient also can increase the dispersion of dried powder in aerosol, and because of its highly water-soluble, and the dissolving that promotes composition to be being deposited on nucleic acid construct and the target film, in the close contact such as host's lung surface film.

The preparation of virus vector

Use Akta FPLC system, the Measles virus carrier is carried out purifying by the size exclusion chromatography method.The damping fluid that is used for chromatography is phosphate buffered saline buffer (PBS).Then, can be with recombinant virus by different paths, for example, nose is interior or oral, is directly used in infection animal.

The preparation of prokaryotic vector

Make bacterial growth in selective medium up to stationary stage of growth (for example, 24 hours in several 1L flasks) 1: 500 inoculation back.By described cell of centrifugation and abandoning supernatant.With cell precipitation washing 5 times, be resuspended in 200ml with 3 liters of PBS then.Prepare described solution and be used for transduction in by all means the body (for example, in the nose or oral).

Embodiment 13

Immunization method

This method is described in the immunization method that uses among subsequently the embodiment.With expressing total length SARS-CoV M, the plasmid of E and S structural protein, virus vector or bacteria carrier immune mouse.Perhaps, with the SARS-CoV-VLPs immune mouse that separates in cell culture medium.With the every fortnight as many as of the plasmid of as many as 100 μ g three times pass through oral, intravenously or intraperitoneal carries out immunity to mouse.In the situation of virus vector, with about 10 ⁷-10 ¹⁰Per two weeks of infectious virus use as many as 3 times.To comprise one or more expression described herein SARS-CoV polypeptide construct 10 ⁵-10 ⁶Protokaryon transmission is used as many as 3 times in per two weeks of carrier.For SARS-CoV-VLPs, per two weeks of the concentration between about 1 μ g and about 10mg are used as many as 3 times.

By the cell-mediated immunne response of one or more following determination and analysis at immunity:

Lymphopoiesis is measured:

With Con A splenocyte (spleenocyte) is carried out pulse, or it is not stimulated, pass through thereby measure ³The propagation that H-thymidine bonded mitogen drives.In order to measure the propagation of antigen-specific, as above cultivate lymphocyte, but with the SARS-CoV recombinant protein of 0.1-10 μ g/ml that has or do not have IL-2, or partially purified SARS-CoV-VLPs carries out burst process to it, and after 7 days, collect.In without any the antigen culture, measure spontaneous propagation.

Lymphokine is replied

In order to measure the generation that stimulates lymphokine, with splenocyte (5-10 * 10 ⁵) with or repeat to cultivate with three times without specific antigens or Con A and carry out incubation.Substratum is to comprise 1% normal mouse serum and antibiotic RPMI.In 37 ℃ of incubations after 48 hours, remove 100 μ l aliquots containig substratum and carry out freezing.Use the HT2 cell that stimulates in response to lymphokine that the lymphokine content in the substratum is measured.

Enzyme linked immunological spot (ELISpot)

Use cell sorter to come purifying CD4 ⁺-CD8 ^-The colony of cell.Carrying out ELISpot measures to estimate the effect of each T-cell part.The mensuration that is used to detect the SARS-CoV cell response comprises T cell proliferation, IL-4 and IFN-γ enzyme linked immunological spot.It is well-known in the art being used for these methods for measuring.

CTLs

Using splenocyte to carry out CTL measures.By routine ⁵¹Chromium-release is measured and is measured the CTL activity.Measuring once more by foregoing stimulation again, CTL replys.To be used as target with EL4 that expresses the antigenic plasmid transfection of SARS and EG7 cell.In order to determine CTL precursor level, we use specific tetramer MHC classification 1 molecule or enzyme linked immunological spot.This technology allows carrying out direct quantitative by the number with the immune T-cell precursors that produces of SARS-CoV immunogen preparation described herein.

As the tumour antigenic SARS protein that rejects

Expressing the rejection of the tumour of virus antigen is mainly regulated by cell-mediated immune responses.Cell-mediated immune responses at the tumour of expressing the coding encoding viral may comprise CD4 ⁺And CD8 ⁺The T cell.

SARS-CoV immunogen preparation described herein is used for inducing protectiveness tumour immunity mouse.With expressing SARS M, the antigenic plasmid of E or S, virus vector or bacteria carrier come intraperitoneal ground immune mouse.The last inoculation is after 7 days, with 1 * 10 ⁵The antigenic B16 of the corresponding SARS of expression, 10 times to the dosage (LD of 50% the animal of causing death ₅₀) dosage to animal carry out in the side of body both sides excite.By making up B16, EL4 and EG7 clone with expressing the antigenic mouse retroviral vector infection of SARS-CoV B16.Determine local tumor growth and mouse survival.In parallel laboratory test, determine whether the CTL activity is relevant with the tumour rejection.In addition, active relevant for making the tumour rejection with immunization protocol, determined of the dependence of tumour rejection activity to the quantity and the inoculation size of dosage.

Embodiment 14

SARS-CoV is immunogenic to be used

Embodiment described isolating SARS-CoV-VLPs prepared product in front maybe can be produced the prepared product of the genetic constructs of SARS-CoV-VLPs, be seeded in the C57blk/6 mouse.The oral ground of described prepared product, intravenously ground or intraperitoneal ground can be inoculated.Specific antibody inducing by enzyme-linked immunosorbent assay (ELISA) in mouse analyzed.Per two weeks are analyzed the serum from the animal that is inoculated, and totally 10 weeks are to determine the development of antibody response.

In other experiment, the prepared product that maybe can produce the genetic constructs of SARS-CoV-VLPs with the isolating SARS-CoV-VLPs prepared product of the increasing amount of describing among the embodiment is in front inoculated animal, thereby whether the size of determining inoculum can determine the level that specific antibody produces.In addition, in order to determine the preferred infection scheme of immunity, with the immunogenic preparation of fixed amount once, carry out intraperitoneal ground for twice, three times or four times and inoculate.The ELISA of use standard measures, and determines in serum and the tiring of the antibody of SARS virus structural protein reaction.

Method described herein allows to inoculate the development that becomes possible strategy when the recombinant vectors of expressing different SARS-CoV antigen proteins makes at multiple antigenic determinant by preparing.In order to test this possibility, with the plasmid inoculation mouse of expressing single and a plurality of SARS-CoV polypeptide.Proteinic each the tiring of antibody of identification SARS virus can be determined by ELISA.Whether this method can induce in the protective immune response at SARS virus at the SARS-CoV antigen of determining single expression is useful.Also this method can be used for the effect of more single antigen method and the effect of a plurality of antigen methods.No matter which kind of method of determining is better, will be appreciated that the immunne response of inducing at any level of SARS virus is useful, and the inducing within the scope of the invention of immunne response of adequately protecting property not.

Embodiment 15

Induce immunne response at SARS-CoV

What will better be accepted is that neutralizing antibody is the important composition of immunne response, and described immunne response provides at the protection that arrives experimenter's pathogenic agent by mucous membrane surface.For example, show, transmit neutralizing antibody, premunition (Kolb etc., 2001) by milk through lactescent recombinant animal.

The antibody that the following examples proof is selected based on external test is useful in animal.In these experiments, with the carrier inoculation mouse of describing among the embodiment in the above that can form VLPs.Particularly, thus to mouse inoculation for several times the inducement efficient valency antibody and get blood at 4-6 after week.To be used for carrying out SARS-CoV and mensuration from the serum of the animal that is inoculated.

Anti--SARS-CoV the antibody of neutralization virus can particularly not have beneficial effect at mucous membrane surface in vivo in tissue culture.For example, can promote virus sweep, virus damage, be suppressed to the transportation or the dysuria with lower abdominal colic of target tissue by complement-mediated with natural viral body bonded antibody, or by the single movability (Robert-Guroff, 2000) that reduces virus of rete malpighii.Each of these effects all reduces the ability that virus causes SARS.

Thereby induce the antibody that to tire with complete S ARS-CoV virosome bonded height with the recombinant vectors inoculation mouse of expressing the SARS-CoV structural protein.In an experiment, collect from by the serum of mice immunized and be used for immunoprecipitation SARS-CoV-VLPs.Thereby carry out western blotting and in sedimentary virosome fraction, detect the SARS-CoV structural protein.Further the concrete SARS-CoV antigen group of check is combined in the disease protection in the non-human primates model, identifies that described concrete SARS-CoV antigen combination induces and natural viral body bonded antibody.

Cytotoxic T lymphocyte (CTLs) is that identification on their surface is incorporated into the cell of kill tumor conversion after the antigen peptide of mixture classification I molecule of main histocompatibility or virus infection.Usually do not excite CTLs with pathogenic agent of normally being killed or their proteinic immunity.

CTL is not being fully understood at the effect in the protective immune response of virus.Yet because CTLs comprises that in removing in the intra-cellular pathogens of broad variety of other coronavirus be important, it is useful stimulating being created in the immunne response of inducing at SARS-CoV of CTL.In addition, in the protection at respiratory disease, CTLs protection respiratory mucous membrane avoids playing an important role in the virus infraction.By from also being given natural animal with protection by the transfer of the CTL of the animal that inoculated.

Embodiment subsequently proves the ability that immunogenicity SARS-CoV preparation described herein excites effective CTL to reply.After with immunogenic SARS-CoV preparation inoculation mouse and macaque, shown the active and high-caliber CTL precursor of high-caliber CTL.In addition, proved that the immunoprotection mouse avoids exciting in order to the tumour of SARS antigen transfection subsequently.

Method described herein, composition and system are the representatives of present embodiment preferred, are exemplary, and are not inclined to as the restriction on the scope of the invention.Variation and other application that those skilled in the art will be made in the present invention spirit scope and be limited by the scope of the invention.Therefore, it will be apparent to one skilled in the art that and not deviate under the scope of the invention and the spiritual prerequisite the present invention as various substitutions and modifications.

Term with below claim and this specification " comprises (comprise) ", " comprising (comprises) " and " comprising (comprising) " be not limited to the invention that requires and get rid of any change or interpolation.On the contrary, term " comprises (comprise) ", " comprising (comprises) " and " comprising (comprising) " mean " comprise, but must not be defined in ".For example, can think accurately to comprise A that the method for B and C, device, molecule or other project comprise A and B.Equally, the method that " comprises A and B ", device, molecule or other project can comprise other step, composition, atom or other project of any number.

When being used for following claim and this specification, " form by ... .. substantially " as for phrase and to mean any composition of behind phrase, listing, and other composition is defined in do not disturb or significantly help in the present invention specific activity or effect for listed composition.Therefore, phrase " basically form " illustrate that the composition of listing is to be required or enforceable, but other composition choose wantonly, and depend on they whether obviously the listed composition of influence activity or act on, can maybe cannot occur.

The reference of quoting in this manual do not show these reference any be that any content of technology before this area or these reference is formed the common or general knowledge for those those of ordinary skills.

Reference:

Armstrong etc., 1984, Nature, 308,751-752

Cavallaro and Monto, 1970, J.Infect.Dis., 122,272-279

Cavanagh etc., 1984, Avian Pathology 13,573-583

Cavanagh，1995，In S.G.Siddell，The coronoviridae.Plenum Press，NewYork，London

Christianson etc., 1989, Arch.Virol., 109,185-196

Delmas and Laude, 1990, J.Virol., 64,5367-5375

Drosten etc., 2003, N.Engl.J.Med., online release

Eljamal etc., 1995, Assignee:Inhale Therapeutics, Patent appl.No.422563FDA, 1996FDA CBER Website.

Godet etc., 1992, Virology, 188,666-675

Grosse etc. 1994, Virology, 202,814-824

Hamre and Prochnow, 1966, Proc.Soc.Exp.Biol.Med., 121,190-193

Hofmann and Wyler, 1987, Schweiz.Arch.Tierheilk., 129,437-442

Holmes etc., 1981, Virology, 115,334-344

Kolb etc., 2001, J Virol., 75,2803-2809

Kozak，1987，Nucleic Acids Res.，15，8125-48.

Ksiazek etc., 2003, N.Engl.J.Med., 348,1947

Lee etc., 2003, N.Engl.J.Med., 348,1986-94.

Liu and Inglis, 1991, Virology 185,911-917

Luytyes etc., 1987, Virology, 161,479-487

MacNaughton at al.，1978，J.Gen.Virol.，39，545-549

MacNaughton etc., 1983, Infect.Immun., 38,419-423

Matsumoto and Kawana, 1992, Kansenshogaku Zasshi, 66,319-326

Niemann and Klenk, 1981, J.Mol.Biol., 153,993-1010

Opstelten etc., 1993, J.Virol., 67,7394-7401

Opstelten etc., 1994, Adv.Exp.Med.Biol., 342,189-195

Opstelten etc., 1995, Adv.Exp.Med.Biol., 380,291-297

Peiris etc., 2003, Lancet, 361,1319

Poutanen etc., 2003, N.Engl.J.Med., 348,1995-2005

Risco etc., 1996, J.Virol., 70,4773-4777

Robert-Guroff，2000，Nat Med.，6，129-130.

Rottier at al.，1981，J.Virol.，40，350-357

Rottier at al.，1986，Biochemistry，25，1335-1339

Sambrook etc., 2001, A laboratory manual

Seo etc., 2000, Virology, 269,183-189

Sturman etc., 1980, J.Virol., 33,449-462

Takamura etc., 2002, Can J Vet Res., 66,278-281.

Takeda etc., 2000, J Virol., 74,6643-6647

Tooze etc., 1987, J.Cell.Biol., 105,1215-1226

Tsang etc., 2003, N.Engl.J.Med., 348,1977-1985

Tyrell etc., 1965, Br.Med.J., 1,1467-1470

Venemma etc., 1990, J.Virol., 64,330-346

Vennema etc., 1991, Virology, 181,327-335

Venemma etc., 1996, EMBO J., 15,2020-2028

Zhang etc., 1994, J Med Virol.44,152-161

Marra etc., 2003, Science, 300,1399-1405

Rota etc., 2003, Science, 300,1394-1399.

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＜213〉sars coronavirus

<400>8

Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Gln Leu Leu

1 5 10 15

Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Ala Trp Ile Met

20 25 30

Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile

35 40 45

Lys Leu Val Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe

50 55 60

Val Leu Ala Val Val Tyr Arg Ile Asn Trp Val Thr Gly Gly Ile Ala

65 70 75 80

Ile Ala Met Ala Cys Ile Val Gly Leu Met Trp Leu Ser Tyr Phe Val

85 90 95

Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn

100 105 110

Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu Arg Gly Thr Ile Val

115 120 125

Thr Arg Pro Leu Met Glu Ser Glu Leu Val Ile Gly Ala Val Ile Ile

130 135 140

Arg Gly His Leu Arg Met Ala Gly His Ser Leu Gly Arg Cys Asp Ile

145 150 155 160

Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser

165 170 175

Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Gly Thr Asp Ser Gly Phe

180 185 190

Ala Ala Tyr Asn Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp

195 200 205

His Ala Gly Ser Asn Asp Asn Ile Ala Leu Leu Val Gln

210 215 220

<210>9

<211>221

<212>PRT

＜213〉sars coronavirus

<400>9

Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Gln Leu Leu

1 5 10 15

Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Ala Trp Ile Met

20 25 30

Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile

35 40 45

Lys Leu Val Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe

50 55 60

Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Val Thr Gly Gly Ile Ala

65 70 75 80

Ile Ala Met Ala Cys Ile Val Gly Leu Met Trp Leu Ser Tyr Phe Val

85 90 95

Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn

100 105 110

Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu Arg Gly Thr Ile Val

115 120 125

Thr Arg Pro Leu Met Glu Ser Glu Leu Val Ile Gly Ala Val Ile Ile

130 135 140

Arg Gly His Leu Arg Met Ala Gly His Ser Leu Gly Arg Cys Asp Ile

145 150 155 160

Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser

165 170 175

Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Gly Thr Asp Ser Gly Phe

180 185 190

Ala Ala Tyr Asn Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp

195 200 205

His Ala Gly Ser Asn Asp Asn Ile Ala Leu Leu Val Gln

210 215 220

<210>10

<211>1255

<212>PRT

＜213〉sars coronavirus

<400>10

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asr Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Lau Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr

1250 1255

<210>11

<211>1255

<212>PRT

＜213〉sars coronavirus

<400>11

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr

1250 1255

<210>12

<211>1255

<212>PRT

＜213〉sars coronavirus

<400>12

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly GIn Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr

1250 1255

<210>13

<211>1255

<212>PRT

＜213〉sars coronavirus

<400>13

Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu

1 5 10 15

Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln

20 25 30

His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45

Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60

Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp Asn Pro Val

65 70 75 80

Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn

85 90 95

Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110

Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125

Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140

Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr

145 150 155 160

Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser

165 170 175

Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190

Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205

Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220

Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

225 230 235 240

Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr

245 250 255

Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285

Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser

325 330 335

Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430

Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445

Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460

Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

465 470 475 480

Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val

485 490 495

Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510

Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540

Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

545 550 555 560

Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys

565 570 575

Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590

Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605

Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620

Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

625 630 635 640

His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile

645 650 655

Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670

Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685

Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700

Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys

705 710 715 720

Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu

725 730 735

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750

Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765

Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780

Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile

785 790 795 800

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met

805 810 815

Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845

Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850 855 860

Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

865 870 875 880

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895

Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910

Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly

915 920 925

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

945 950 955 960

Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp

965 970 975

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1010 1015 1020

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His

1025 1030 1035 1040

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

1045 1050 1055

Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

1060 1065 1070

Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

1075 1080 1085

Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1090 1095 1100

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr

1105 1110 1115 1120

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1125 1130 1135

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1140 1145 1150

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1155 1160 1165

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1170 1175 1180

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu

1185 1190 1195 1200

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu

1205 1210 1215

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

1220 1225 1230

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245

Gly Val Lys Leu His Tyr Thr

1250 1255

<210>14

<211>29751

<2l2>DNA

＜213〉sars coronavirus

<400>14

atattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 60

ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 120

gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 180

tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 240

gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 300

cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 360

gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 420

ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 480

cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 540

gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 600

gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 660

ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 720

cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 780

ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 840

ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 900

tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 960

gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1020

acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1080

tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1140

actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1200

aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1260

acgtgcgact ttctgaaagc cacttgtgaa cattgtggca ctgaaaattt agttattgaa 1320

ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1380

tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1440

attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1500

tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1560

tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1620

atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1680

gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1740

agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1800

aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1860

ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1920

gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1980

atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2040

aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2100

ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2160

gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2220

attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2280

gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2340

gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag gtgaagtctt catcgctcaa 2400

agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2460

cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2520

tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2580

ttcacaaatg gagctatcgt tggcacacca gtctgtgtaa atggcctcat gctcttagag 2640

attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2700

tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2760

gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2820

gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2880

gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2940

aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3000

ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3060

gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3120

acagaggatg attatcaagg tctccctctg gaatttggtg cctcagctga aacagttcga 3180

gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3240

ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3300

actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3360

atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3420

ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3480

ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3540

ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3600

tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3660

ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3720

attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3780

aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3840

gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3900

gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3960

gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4020

tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4080

acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4140

ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4200

tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4260

ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4320

gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4380

gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4440

gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4500

aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4560

tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4620

gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4680

tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4740

tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4800

cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4860

ctaaagagtc tcttatccct gcgggaggtt aagactataa aagtgttcac aactgtggac 4920

aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4980

ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5040

aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5100

catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5160

tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5220

ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5280

caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5340

gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5400

ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5460

ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5520

tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5580

tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5640

ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5700

tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5760

atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5820

accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5880

ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5940

ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6000

aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6060

tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6120

tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6180

caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6240

acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6300

atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6360

accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6420

atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6480

atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6540

gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6600

agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6660

tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6720

ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6780

acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6840

aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6900

ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6960

aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7020

gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7080

gactcccttg attcttatcc agctcttgaa actattcagg tgacgatttc atcgtacaag 7140

ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7200

aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7260

agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7320

cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7380

agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7440

aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7500

gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7560

gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7620

cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7680

ctgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7740

catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7800

ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7860

tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagct 7920

cttgtatcag acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7980

gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8040

gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8100

gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8160

aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8220

acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8280

gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8340

aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtagtgc tgccaagaag 8400

aacaacatac cttttagact aacttgtgct acaactagac aggttgtcaa tgtcataact 8460

actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8520

gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8580

ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8640

gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8700

gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8760

gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga 8820

gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8880

tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8940

gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9000

actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9060

cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9120

gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9180

atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9240

ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9300

caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9360

ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9420

catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9480

ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9540

ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9600

ctgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9660

ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9720

gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9780

gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9840

tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9900

aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9960

tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa 10020

gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg 10080

gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct 10140

aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat 10200

gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat 10260

acttctaacc ctaagacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt 10320

tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct 10380

aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt 10440

gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac 10500

gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag 10560

gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt 10620

atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt 10680

gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct 10740

ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg 10800

cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca 10860

ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt 10920

gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt 10980

caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact 11040

cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc 11100

ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg 11160

cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct 11220

ggttataggc ttaaggattg tgttatgtat gcttcagctt tagttttgct tattctcatg 11280

acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt 11340

acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc 11400

ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct 11460

agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc 11520

ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc 11580

cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc 11640

tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt 11700

gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt 11760

gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt 11820

cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac 11880

aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg 11940

tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc 12000

gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc 12060

gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc 12120

gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12180

gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12240

gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12300

atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12360

tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12420

gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12480

tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12540

atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12600

gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12660

gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12720

aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12780

ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12840

gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12900

aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12960

aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13020

cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13080

aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13140

atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13200

catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13260

tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13320

tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13380

gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13440

caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaagttgctg 13500

gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13560

atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13620

agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13680

ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13740

tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13800

aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13860

acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13920

aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13980

tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14040

aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14100

tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14160

cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14220

accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14280

ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14340

caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14400

ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14460

cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14520

ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14580

atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14640

tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14700

aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14760

gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14820

atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14880

tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14940

aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15000

ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15060

gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15120

gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15180

ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15240

gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15300

cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15360

gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15420

atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15480

taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15540

aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15600

agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15660

tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15720

cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15780

accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15840

atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15900

tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15960

ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16020

atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16080

ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16140

tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16200

cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16260

accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16320

ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16380

gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16440

tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16500

gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16560

ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16620

ccactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16680

ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16740

aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16800

gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16860

taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16920

tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16980

tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17040

ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17100

cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17160

gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17220

tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17280

tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17340

gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17400

tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17460

taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17520

tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17580

tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17640

aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17700

tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17760

ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17820

cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17880

ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17940

taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18000

gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18060

taaagttcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18120

accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18180

atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18240

tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18300

tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18360

cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18420

cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18480

gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18540

agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctgtgtg 18600

acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18660

tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18720

gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18780

gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18840

attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18900

aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gttcttcatg 18960

acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19020

acgatgctca gccatgtagt gacaaagctt acaaaataga ggaactcttc tattcttatg 19080

ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19140

gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agtcttgtca aacttgaact 19200

taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19260

tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19320

cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19380

ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19440

accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19500

acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19560

atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19620

tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19680

aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19740

aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19800

taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19860

tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19920

atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19980

cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20040

gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20100

gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20160

agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20220

gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20280

aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20340

aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20400

aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20460

agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20520

atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20580

aactacaagc aagtcaagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20640

aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20700

aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20760

ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20820

ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20880

cagatcttaa tgacttcgtc tccgacgcag attctacttt aattggagac tgtgcaacag 20940

tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21000

atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21060

agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21120

ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21180

atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21240

aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21300

agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21360

ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21420

gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca 21480

actaaacgaa catgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg 21540

accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta 21600

tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg 21660

atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg 21720

gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg 21780

ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta 21840

ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt 21900

tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat 21960

ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag 22020

gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt 22080

ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga 22140

aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag 22200

ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt 22260

taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg 22320

attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca 22380

aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc 22440

ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg 22500

tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca 22560

actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc 22620

tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa 22680

tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca 22740

tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata 22800

attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta 22860

atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc 22920

cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg 22980

tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca 23040

ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg 23100

tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg 23160

atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgcg 23220

cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc 23280

tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac 23340

cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta 23400

taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt 23460

gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt 23520

atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac 23580

ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct 23640

ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc 23700

aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg 23760

atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga 23820

aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga 23880

ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga 23940

agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt 24000

tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg 24060

ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc 24120

aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg 24180

ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc 24240

aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga 24300

atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa 24360

gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca 24420

ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg 24480

ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg 24540

gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag 24600

cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact 24660

tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt 24720

ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa 24780

ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca 24840

acacagttta tgatcctctg caacctgagc ttgactcatt caaagaagag ctggacaagt 24900

acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt 24960

ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg 25020

aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt 25080

atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt 25140

gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca 25200

agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataaa 25260

cgaacttatg gatttgttta tgagattttt tactcttaga tcaattactg cacagccagt 25320

aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25380

agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25440

cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25500

gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25560

tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25620

caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25680

attactttat gatgccaact actttgtttg ctggcacaca cataactatg actactgtat 25740

accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25800

aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25860

agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25920

aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25980

agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26040

aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga 26100

aagtgagtac gaacttatgt actcattcgt ttcggaagaa acaggtacgt taatagttaa 26160

tagcgtactt ctttttcttg ctttcgtggt attcttgcta gtcacactag ccatccttac 26220

tgcgcttcga ttgtgtgcgt actgctgcaa tattgttaac gtgagtttag taaaaccaac 26280

ggtttacgtc tactcgcgtg ttaaaaatct gaactcttct gaaggagttc ctgatcttct 26340

ggtctaaacg aactaactat tattattatt ctgtttggaa ctttaacatt gcttatcatg 26400

gcagacaacg gtactattac cgttgaggag cttaaacaac tcctggaaca atggaaccta 26460

gtaataggtt tcctattcct agcctggatt atgttactac aatttgccta ttctaatcgg 26520

aacaggtttt tgtacataat aaagcttgtt ttcctctggc tcttgtggcc agtaacactt 26580

gcttgttttg tgcttgctgc tgtctacaga attaattggg tgactggcgg gattgcgatt 26640

gcaatggctt gtattgtagg cttgatgtgg cttagctact tcgttgcttc cttcaggctg 26700

tttgctcgta cccgctcaat gtggtcattc aacccagaaa caaacattct tctcaatgtg 26760

cctctccggg ggacaattgt gaccagaccg ctcatggaaa gtgaacttgt cattggtgct 26820

gtgatcattc gtggtcactt gcgaatggcc ggacactccc tagggcgctg tgacattaag 26880

gacctgccaa aagagatcac tgtggctaca tcacgaacgc tttcttatta caaattagga 26940

gcgtcgcagc gtgtaggcac tgattcaggt tttgctgcat acaaccgcta ccgtattgga 27000

aactataaat taaatacaga ccacgccggt agcaacgaca atattgcttt gctagtacag 27060

taagtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat 27120

tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat 27180

agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga 27240

acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga 27300

ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac 27360

tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg 27420

ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg 27480

gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac 27540

aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat 27600

ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga 27660

cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt 27720

ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat 27780

gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca 27840

gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg 27900

gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat 27960

ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg 28020

gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta 28080

gagacgtact tgttgtttta aataaacgaa caaattaaaa tgtctgataa tggaccccaa 28140

tcaaaccaac gtagtgcccc ccgcattaca tttggtggac ccacagattc aactgacaat 28200

aaccagaatg gaggacgcaa tggggcaagg ccaaaacagc gccgacccca aggtttaccc 28260

aataatactg cgtcttggtt cacagctctc actcagcatg gcaaggagga acttagattc 28320

cctcgaggcc agggcgttcc aatcaacacc aatagtggtc cagatgacca aattggctac 28380

taccgaagag ctacccgacg agttcgtggt ggtgacggca aaatgaaaga gctcagcccc 28440

agatggtact tctattacct aggaactggc ccagaagctt cacttcccta cggcgctaac 28500

aaagaaggca tcgtatgggt tgcaactgag ggagccttga atacacccaa agaccacatt 28560

ggcacccgca atcctaataa caatgctgcc accgtgctac aacttcctca aggaacaaca 28620

ttgccaaaag gcttctacgc agagggaagc agaggcggca gtcaagcctc ttctcgctcc 28680

tcatcacgta gtcgcggtaa ttcaagaaat tcaactcctg gcagcagtag gggaaattct 28740

cctgctcgaa tggctagcgg aggtggtgaa actgccctcg cgctattgct gctagacaga 28800

ttgaaccagc ttgagagcaa agtttctggt aaaggccaac aacaacaagg ccaaactgtc 28860

actaagaaat ctgctgctga ggcatctaaa aagcctcgcc aaaaacgtac tgccacaaaa 28920

cagtacaacg tcactcaagc atttgggaga cgtggtccag aacaaaccca aggaaatttc 28980

ggggaccaag acctaatcag acaaggaact gattacaaac attggccgca aattgcacaa 29040

tttgctccaa gtgcctctgc attctttgga atgtaacgca ttggcatgga agtcacaact 29100

tcgggaacat ggctgactta tcatggagcc attaaattgg atgacaaaga tccacaattc 29160

aaagacaacg tcatactgct gaacaagcac attgacgcat acaaaacatt cccaccaaca 29220

gagcctaaaa aggacaaaaa gaaaaagact gatgaagctc agcctttgcc gcagagacaa 29280

aagaagcagc ccactgtgac tcttcttcct gcggctgaca tggatgattt ctccagacaa 29340

cttcaaaatt ccatgagtgg agcttctgct gattcaactc aggcataaac actcatgatg 29400

accacacaag gcagatgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29460

tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29520

atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29580

cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29640

ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29700

attttaatag cttcttagga gaatgacaaa aaaaaaaaaa aaaaaaaaaa a 29751

<210>15

<211>29727

<212>DNA

＜213〉sars coronavirus

<400>15

atattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 60

ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 120

gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 180

tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 240

gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 300

cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 360

gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 420

ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 480

cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 540

gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 600

gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 660

ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 720

cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 780

ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 840

ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 900

tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 960

gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1020

acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1080

tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1140

actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1200

aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1260

acgtgcgact ttctgaaagc cacttgtgaa cattgtggca ctgaaaattt agttattgaa 1320

ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1380

tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1440

attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1500

tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1560

tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1620

atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1680

gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1740

agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1800

aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1860

ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1920

gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1980

atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2040

aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2100

ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2160

gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2220

attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2280

gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2340

gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag gtgaagtctt catcgctcaa 2400

agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2460

cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2520

tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2580

ttcacaaatg gagctatcgt tggcacacca gtctgtgtaa atggcctcat gctcttagag 2640

attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2700

tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2760

gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2820

gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2880

gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2940

aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3000

ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3060

gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3120

acagaggatg attatcaagg tctccctctg gaatttggtg cctcagctga aacagttcga 3180

gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3240

ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3300

actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3360

atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3420

ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3480

ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3540

ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3600

tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3660

ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3720

attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3780

aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3840

gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3900

gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3960

gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4020

tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4080

acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4140

ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4200

tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4260

ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4320

gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4380

gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4440

gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4500

aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4560

tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4620

gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4680

tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4740

tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4800

cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4860

ctaaagagtc tcttatccct gcggcaggtt aagactataa aagtgttcac aactgtggac 4920

aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4980

ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5040

aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5100

catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5160

tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5220

ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5280

caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5340

gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5400

ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5460

ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5520

tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5580

tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5640

ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5700

tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5760

atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5820

accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5880

ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5940

ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6000

aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6060

tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6120

tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6180

caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6240

acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6300

atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6360

accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6420

atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6480

atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6540

gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6600

agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6660

tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6720

ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6780

acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6840

aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6900

ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6960

aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7020

gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7080

gactcccttg attcttatcc agctcttgaa accattcagg tgacgatttc atcgtacaag 7140

ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7200

aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7260

agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7320

cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7380

agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7440

aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7500

gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7560

gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7620

cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7680

gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7740

catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7800

ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7860

tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagtt 7920

cttgtatcag acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7980

gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8040

gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8100

gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8160

aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8220

acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8280

gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8340

aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtagtgc tgccaagaag 8400

aacaacatac cttttagact aacttgtgct acaactagac aggttgtcaa tgtcataact 8460

actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8520

gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8580

ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8640

gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8700

gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8760

gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga 8820

gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8880

tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8940

gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9000

actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9060

cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9120

gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9180

atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9240

ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9300

caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9360

ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9420

catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9480

ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9540

ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9600

gtgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9660

ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9720

gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9780

gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9840

tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9900

aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9960

tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa 10020

gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg 10080

gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct 10140

aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat 10200

gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat 10260

acttctaacc ctaacacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt 10320

tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct 10380

aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt 10440

gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac 10500

gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag 10560

gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt 10620

atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt 10680

gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct 10740

ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg 10800

cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca 10860

ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt 10920

gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt 10980

caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact 11040

cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc 11100

ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg 11160

cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct 11220

ggttataggc ttaaggattg tgttatgtat gcttcagctt tagttttgct tattctcatg 11280

acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt 11340

acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc 11400

ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct 11460

agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc 11520

ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc 11580

cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc 11640

tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt 11700

gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt 11760

gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt 11820

cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac 11880

aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg 11940

tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc 12000

gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc 12060

gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc 12120

gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12180

gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12240

gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12300

atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12360

tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12420

gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12480

tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12540

atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12600

gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12660

gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12720

aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12780

ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12840

gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12900

aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12960

aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13020

cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13080

aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13140

atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13200

catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13260

tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13320

tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13380

gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13440

caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaagttgctg 13500

gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13560

atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13620

agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13680

ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13740

tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13800

aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13860

acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13920

aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13980

tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14040

aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14100

tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14160

cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14220

accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14280

ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14340

caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14400

ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14460

cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14520

ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14580

atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14640

tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14700

aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14760

gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14820

atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14880

tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14940

aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15000

ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15060

gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15120

gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15180

ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15240

gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15300

cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15360

gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15420

atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15480

taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15540

aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15600

agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15660

tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15720

cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15780

accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15840

atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15900

tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15960

ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16020

atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16080

ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16140

tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16200

cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16260

accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16320

ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16380

gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16440

tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16500

gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16560

ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16620

ctactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16680

ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16740

aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16800

gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16860

taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16920

tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16980

tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17040

ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17100

cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17160

gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17220

tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17280

tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17340

gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17400

tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17460

taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17520

tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17580

tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17640

aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17700

tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17760

ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17820

cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17880

ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17940

taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18000

gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18060

taaagttcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18120

accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18180

atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18240

tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18300

tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18360

cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18420

cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18480

gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18540

agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctgtgtg 18600

acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18660

tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18720

gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18780

gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18840

attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18900

aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gttcttcatg 18960

acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19020

acgatgctca gccatgtagt gacaaagctt acaaaataga ggagctcttc tattcttatg 19080

ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19140

gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agtcttgtca aacttgaact 19200

taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19260

tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19320

cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19380

ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19440

accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19500

acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19560

atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19620

tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19680

aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19740

aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19800

taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19860

tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19920

atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19980

cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20040

gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20100

gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20160

agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20220

gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20280

aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20340

aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20400

aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20460

agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20520

atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20580

aactacaagc aagtcaagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20640

aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20700

aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20760

ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20820

ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20880

cagatcttaa tgacttcgtc tccgacgcag attctacttt aattggagac tgtgcaacag 20940

tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21000

atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21060

agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21120

ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21180

atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21240

aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21300

agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21360

ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21420

gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca 21480

actaaacgaa catgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg 21540

accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta 21600

tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg 21660

atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg 21720

gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg 21780

ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta 21840

ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt 21900

tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat 21960

ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag 22020

gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt 22080

ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga 22140

aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag 22200

ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt 22260

taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg 22320

attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca 22380

aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc 22440

ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg 22500

tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca 22560

actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc 22620

tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa 22680

tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca 22740

tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata 22800

attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta 22860

atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc 22920

cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg 22980

tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca 23040

ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg 23100

tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg 23160

atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct 23220

cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc 23280

tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac 23340

cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta 23400

taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt 23460

gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt 23520

atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac 23580

ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct 23640

ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc 23700

aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg 23760

atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga 23820

aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga 23880

ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga 23940

agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt 24000

tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg 24060

ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc 24120

aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg 24180

ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc 24240

aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga 24300

atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa 24360

gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca 24420

ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg 24480

ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg 24540

gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag 24600

cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact 24660

tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt 24720

ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa 24780

ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca 24840

acacagttta tgatcctctg caacctgagc tcgactcatt caaagaagag ctggacaagt 24900

acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt 24960

ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg 25020

aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt 25080

atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt 25140

gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca 25200

agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataaa 25260

cgaacttatg gatttgttta tgagattttt tactcttgga tcaattactg cacagccagt 25320

aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25380

agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25440

cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25500

gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25560

tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25620

caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25680

attactttat gatgccaact actttgtttg ctggcacaca cataactatg actactgtat 25740

accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25800

aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25860

agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25920

aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25980

agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26040

aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga 26100

aagtgagtac gaacttatgt actcattcgt ttcggaagaa acaggtacgt taatagttaa 26160

tagcgtactt ctttttcttg ctttcgtggt attcttgcta gtcacactag ccatccttac 26220

tgcgcttcga ttgtgtgcgt actgctgcaa tattgttaac gtgagtttag taaaaccaac 26280

ggtttacgtc tactcgcgtg ttaaaaatct gaactcttct gaaggagttc ctgatcttct 26340

ggtctaaacg aactaactat tattattatt ctgtttggaa ctttaacatt gcttatcatg 26400

gcagacaacg gtactattac cgttgaggag cttaaacaac tcctggaaca atggaaccta 26460

gtaataggtt tcctattcct agcctggatt atgttactac aatttgccta ttctaatcgg 26520

aacaggtttt tgtacataat aaagcttgtt ttcctctggc tcttgtggcc agtaacactt 26580

gcttgttttg tgcttgctgc tgtctacaga attaattggg tgactggcgg gattgcgatt 26640

gcaatggctt gtattgtagg cttgatgtgg cttagctact tcgttgcttc cttcaggctg 26700

tttgctcgta cccgctcaat gtggtcattc aacccagaaa caaacattct tctcaatgtg 26760

cctctccggg ggacaattgt gaccagaccg ctcatggaaa gtgaacttgt cattggtgct 26820

gtgatcattc gtggtcactt gcgaatggcc ggacaccccc tagggcgctg tgacattaag 26880

gacctgccaa aagagatcac tgtggctaca tcacgaacgc tttcttatta caaattagga 26940

gcgtcgcagc gtgtaggcac tgattcaggt tttgctgcat acaaccgcta ccgtattgga 27000

aactataaat taaatacaga ccacgccggt agcaacgaca atattgcttt gctagtacag 27060

taagtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat 27120

tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat 27180

agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga 27240

acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga 27300

ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac 27360

tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg 27420

ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg 27480

gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac 27540

aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat 27600

ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga 27660

cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt 27720

ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat 27780

gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca 27840

gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg 27900

gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat 27960

ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg 28020

gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta 28080

gagacgtact tgttgtttta aataaacgaa caaattaaaa tgtctgataa tggaccccaa 28140

tcaaaccaac gtagtgcccc ccgcattaca tttggtggac ccacagattc aactgacaat 28200

aaccagaatg gaggacgcaa tggggcaagg ccaaaacagc gccgacccca aggtttaccc 28260

aataatactg cgtcttggtt cacagctctc actcagcatg gcaaggagga acttagattc 28320

cctcgaggcc agggcgttcc aatcaacacc aatagtggtc cagatgacca aattggctac 28380

taccgaagag ctacccgacg agttcgtggt ggtgacggca aaatgaaaga gctcagcccc 28440

agatggtact tctattacct aggaactggc ccagaagctt cacttcccta cggcgctaac 28500

aaagaaggca tcgtatgggt tgcaactgag ggagccttga atacacccaa agaccacatt 28560

ggcacccgca atcctaataa caatgctgcc accgtgctac aacttcctca aggaacaaca 28620

ttgccaaaag gcttctacgc agagggaagc agaggcggca gtcaagcctc ttctcgctcc 28680

tcatcacgta gtcgcggtaa ttcaagaaat tcaactcctg gcagcagtag gggaaattct 28740

cctgctcgaa tggctagcgg aggtggtgaa actgccctcg cgctattgct gctagacaga 28800

ttgaaccagc ttgagagcaa agtttctggt aaaggccaac aacaacaagg ccaaactgtc 28860

actaagaaat ctgctgctga ggcatctaaa aagcctcgcc aaaaacgtac tgccacaaaa 28920

cagtacaacg tcactcaagc atttgggaga cgtggtccag aacaaaccca aggaaatttc 28980

ggggaccaag acctaatcag acaaggaact gattacaaac attggccgca aattgcacaa 29040

tttgctccaa gtgcctctgc attctttgga atgtcacgca ttggcatgga agtcacacct 29100

tcgggaacat ggctgactta tcatggagcc attaaattgg atgacaaaga tccacaattc 29160

aaagacaacg tcatactgct gaacaagcac attgacgcat acaaaacatt cccaccaaca 29220

gagcctaaaa aggacaaaaa gaaaaagact gatgaagctc agcctttgcc gcagagacaa 29280

aagaagcagc ccactgtgac tcttcttcct gcggctgaca tggatgattt ctccagacaa 29340

cttcaaaatt ccatgagtgg agcttctgct gattcaactc aggcataaac actcatgatg 29400

accacacaag gcagatgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29460

tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29520

atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29580

cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29640

ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29700

attttaatag cttcttagga gaatgac 29727

<210>16

<211>29742

<212>DNA

＜213〉sars coronavirus

<400>16

atattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 60

ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 120

gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 180

tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 240

gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 300

cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 360

gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 420

ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 480

cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 540

gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 600

gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 660

ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 720

cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 780

ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 840

ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 900

tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 960

gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1020

acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1080

tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1140

actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1200

aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1260

acgtgcgact ttctgaaagc cacttgtgaa cattgtggca ctgaaaattt agttattgaa 1320

ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1380

tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1440

attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1500

tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1560

tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1620

atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1680

gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1740

agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1800

aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1860

ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1920

gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1980

atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2040

aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2100

ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2160

gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2220

attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2280

gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2340

gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag gtgaagtctt catcgctcaa 2400

agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2460

cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2520

tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2580

ttcacaaatg gagctatcgt cggcacacca gtctgtgtaa atggcctcat gctcttagag 2640

attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2700

tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2760

gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2820

gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2880

gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2940

aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3000

ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3060

gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3120

acagaggatg attatcaagg tctccctctg gaatttggtg cctcagctga aacagttcga 3180

gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3240

ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3300

actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3360

atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3420

ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3480

ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3540

ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3600

tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3660

ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3720

attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3780

aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3840

gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3900

gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3960

gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4020

tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4080

acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4140

ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4200

tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4260

ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4320

gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4380

gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4440

gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4500

aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4560

tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4620

gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4680

tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4740

tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4800

cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4860

ctaaagagtc tcttatccct gcgggaggtt aagactataa aagtgttcac aactgtggac 4920

aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4980

ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5040

aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5100

catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5160

tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5220

ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5280

caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5340

gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5400

ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5460

ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5520

tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5580

tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5640

ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5700

tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5760

atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5820

accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5880

ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5940

ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6000

aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6060

tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6120

tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6180

caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6240

acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6300

atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6360

accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6420

atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6480

atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6540

gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6600

agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6660

tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6720

ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6780

acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6840

aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6900

ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6960

aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7020

gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7080

gactcccttg attcttatcc agctcttgaa accattcagg tgacgatttc atcgtacaag 7140

ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7200

aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7260

agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7320

cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7380

agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7440

aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7500

gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7560

gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7620

cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7680

gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7740

catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7800

ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7860

tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagct 7920

cttgtatcaa acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7980

gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8040

gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8100

gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8160

aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8220

acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8280

gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8340

aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtactgc tgccaagaag 8400

aacaacatac cttttacact aacttgtgct acaactagac aggttgtcaa tgtcataact 8460

actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8520

gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8580

ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8640

gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8700

gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8760

gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga 8820

gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8880

tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8940

gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9000

actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9060

cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9120

gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9180

atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9240

ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9300

caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9360

ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9420

catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9480

ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9540

ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9600

gtgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9660

ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9720

gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9780

gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9840

tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9900

aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9960

tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa 10020

gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg 10080

gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct 10140

aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat 10200

gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat 10260

acttctaacc ctaagacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt 10320

tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct 10380

aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt 10440

gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac 10500

gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag 10560

gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt 10620

atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt 10680

gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct 10740

ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg 10800

cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca 10860

ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt 10920

gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt 10980

caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact 11040

cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc 11100

ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg 11160

cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct 11220

ggttataggc ttaaggattg tgttatgtat gcttcagctc tagttttgct tattctcatg 11280

acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt 11340

acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc 11400

ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct 11460

agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc 11520

ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc 11580

cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc 11640

tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt 11700

gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt 11760

gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt 11820

cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac 11880

aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg 11940

tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc 12000

gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc 12060

gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc 12120

gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12180

gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12240

gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12300

atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12360

tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12420

gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12480

tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12540

atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12600

gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12660

gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12720

aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12780

ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12840

gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12900

aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12960

aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13020

cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13080

aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13140

atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13200

catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13260

tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13320

tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13380

gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13440

caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaaagtgctg 13500

gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13560

atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13620

agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13680

ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13740

tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13800

aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13860

acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13920

aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13980

tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14040

aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14100

tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14160

cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14220

accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14280

ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14340

caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14400

ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14460

cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14520

ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14580

atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14640

tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14700

aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14760

gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14820

atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14880

tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14940

aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15000

ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15060

gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15120

gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15180

ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15240

gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15300

cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15360

gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15420

atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15480

taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15540

aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15600

agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15660

tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15720

cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15780

accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15840

atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15900

tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15960

ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16020

atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16080

ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16140

tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16200

cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16260

accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16320

ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16380

gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16440

tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16500

gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16560

ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16620

ccactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16680

ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16740

aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16800

gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16860

taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16920

tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16980

tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17040

ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17100

cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17160

gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17220

tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17280

tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17340

gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17400

tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17460

taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17520

tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17580

tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17640

aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17700

tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17760

ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17820

cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17880

ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17940

taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18000

gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18060

taaaattcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18120

accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18180

atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18240

tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18300

tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18360

cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18420

cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18480

gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18540

agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctggttg 18600

acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18660

tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18720

gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18780

gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18840

attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18900

aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gttcttcatg 18960

acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19020

acgatgctca gccatgtagt gacaaagctt acaaaataga ggaactcttc tattcttatg 19080

ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19140

gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agtcttgtca aacttgaact 19200

taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19260

tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19320

cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19380

ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19440

accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19500

acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19560

atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19620

tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19680

aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19740

aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19800

taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19860

tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19920

atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19980

cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20040

gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20100

gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20160

agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20220

gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20280

aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20340

aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20400

aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20460

agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20520

atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20580

aactacaagc aagtcaagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20640

aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20700

aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20760

ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20820

ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20880

cagatcttaa tgacttcgtc tccgacgcag attctacttt aattggagac tgtgcaacag 20940

tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21000

atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21060

agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21120

ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21180

atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21240

aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21300

agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21360

ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21420

gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca 21480

actaaacgaa catgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg 21540

accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta 21600

tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg 21660

atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg 21720

gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg 21780

ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta 21840

ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt 21900

tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat 21960

ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag 22020

gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt 22080

ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga 22140

aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag 22200

ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt 22260

taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg 22320

attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca 22380

aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc 22440

ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg 22500

tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca 22560

actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc 22620

tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa 22680

tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca 22740

tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata 22800

attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta 22860

atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc 22920

cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg 22980

tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca 23040

ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg 23100

tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg 23160

atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct 23220

cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc 23280

tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac 23340

cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta 23400

taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt 23460

gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt 23520

atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac 23580

ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct 23640

ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc 23700

aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg 23760

atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga 23820

aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga 23880

ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga 23940

agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt 24000

tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg 24060

ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc 24120

aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg 24180

ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc 24240

aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga 24300

atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa 24360

gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca 24420

ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg 24480

ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg 24540

gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag 24600

cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact 24660

tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt 24720

ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa 24780

ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca 24840

acacagttta tgatcctctg caacctgagc ttgactcatt caaagaagag ctggacaagt 24900

acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt 24960

ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg 25020

aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt 25080

atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt 25140

gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca 25200

agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataaa 25260

cgaacttatg gatttgttta tgagattttt tactcttgga tcaattactg cacagccagt 25320

aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25380

agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25440

cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25500

gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25560

tgcaggtaag gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25620

caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25680

attactttat catgccaact actttgtttg ctggcacaca cataactatg actactgtat 25740

accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25800

aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25860

agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25920

aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25980

agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26040

aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga 26100

aagtgagtac gaacttatgt actcattcgt ttcggaagaa acaggtacgt taatagttaa 26160

tagcgtactt ctttttcttg ctttcgtggt attcttgcta gtcacactag ccatccttac 26220

tgcgcttcga ttgtgtgcgt actgctgcaa tattgttaac gtgagtttag taaaaccaac 26280

ggtttacgtc tactcgcgtg ttaaaaatct gaactcttct gaaggagttc ctgatcttct 26340

ggtctaaacg aactaactat tattattatt ctgtttggaa ctttaacatt gcttatcatg 26400

gcagacaacg gtactattac cgttgaggag cttaaacaac tcctggaaca atggaaccta 26460

gtaataggtt tcctattcct agcctggatt atgttactac aatttgccta ttctaatcgg 26520

aacaggtttt tgtacataat aaagcttgtt ttcctctggc tcttgtggcc agtaacactt 26580

gcttgttttg tgcttgctgt tgtctacaga attaattggg tgactggcgg gattgcgatt 26640

gcaatggctt gtattgtagg cttgatgtgg cttagctact tcgttgcttc cttcaggctg 26700

tttgctcgta cccgctcaat gtggtcattc aacccagaaa caaacattct tctcaatgtg 26760

cctctccggg ggacaattgt gaccagaccg ctcatggaaa gtgaacttgt cattggtgct 26820

gtgatcattc gtggtcactt gcgaatggcc ggacactccc tagggcgctg tgacattaag 26880

gacctgccaa aagagatcac tgtggctaca tcacgaacgc tttcttatta caaattagga 26940

gcgtcgcagc gtgtaggcac tgattcaggt tttgctgcat acaaccgcta ccgtattgga 27000

aactataaat taaatacaga ccacgccggt agcaacgaca atattgcttt gctagtacag 27060

taagtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat 27120

tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat 27180

agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga 27240

acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga 27300

ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac 27360

tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg 27420

ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg 27480

gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac 27540

aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat 27600

ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga 27660

cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt 27720

ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat 27780

gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca 27840

gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg 27900

gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat 27960

ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg 28020

gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta 28080

gagacgtact tgttgtttta aataaacgaa caaattaaaa tgtctgataa tggaccccaa 28140

tcaaaccaac gtagtgcccc ccgcattaca tttggtggac ccacagattc aactgacaat 28200

aaccagaatg gaggacgcaa tggggcaagg ccaaaacagc gccgacccca aggtttaccc 28260

aataatactg cgtcttggtt cacagctctc actcagcatg gcaaggagga acttagattc 28320

cctcgaggcc agggcgttcc aatcaacacc aatagtggtc cagatgacca aattggctac 28380

taccgaagag ctacccgacg agttcgtggt ggtgacggca aaatgaaaga gctcagcccc 28440

agatggtact tctattacct aggaactggc ccagaagctt cacttcccta cggcgctaac 28500

aaagaaggca tcgtatgggt tgcaactgag ggagccttga atacacccaa agaccacatt 28560

ggcacccgca atcctaataa caatgctgcc accgtgctac aacttcctca aggaacaaca 28620

ttgccaaaag gcttctacgc agagggaagc agaggcggca gtcaagcctc ttctcgctcc 28680

tcatcacgta gtcgcggtaa ttcaagaaat tcaactcctg gcagcagtag gggaaattct 28740

cctgctcgaa tggctagcgg aggtggtgaa actgccctcg cgctattgct gctagacaga 28800

ttgaaccagc ttgagagcaa agtttctggt aaaggccaac aacaacaagg ccaaactgtc 28860

actaagaaat ctgctgctga ggcatctaaa aagcctcgcc aaaaacgtac tgccacaaaa 28920

cagtacaacg tcactcaagc atttgggaga cgtggtccag aacaaaccca aggaaatttc 28980

ggggaccaag acctaatcag acaaggaact gattacaaac attggccgca aattgcacaa 29040

tttgctccaa gtgcctctgc attctttgga atgtcacgca ttggcatgga agtcacacct 29100

tcgggaacat ggctgactta tcatggagcc attaaattgg atgacaaaga tccacaattc 29160

aaagacaacg tcatactgct gaacaagcac attgacgcat acaaaacatt cccaccaaca 29220

gagcctaaaa aggacaaaaa gaaaaagact gatgaagctc agcctttgcc gcagagacaa 29280

aagaagcagc ccactgtgac tcttcttcct gcggctgaca tggatgattt ctccagacaa 29340

cttcaaaatt ccatgagtgg agcttctgct gattcaactc aggcataaac actcatgatg 29400

accacacaag gcagatgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29460

tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29520

atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29580

cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29640

ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29700

attttaatag cttcttagga gaatgacaaa aaaaaaaaaa aa 29742

<210>17

<211>29736

<212>DNA

＜213〉sars coronavirus

<400>17

ctacccagga aaagccaacc aacctcgatc tcttgtagat ctgttctcta aacgaacttt 60

aaaatctgtg tagctgtcgc tcggctgcat gcctagtgca cctacgcagt ataaacaata 120

ataaatttta ctgtcgttga caagaaacga gtaactcgtc cctcttctgc agactgctta 180

cggtttcgtc cgtgttgcag tcgatcatca gcatacctag gtttcgtccg ggtgtgaccg 240

aaaggtaaga tggagagcct tgttcttggt gtcaacgaga aaacacacgt ccaactcagt 300

ttgcctgtcc ttcaggttag agacgtgcta gtgcgtggct tcggggactc tgtggaagag 360

gccctatcgg aggcacgtga acacctcaaa aatggcactt gtggtctagt agagctggaa 420

aaaggcgtac tgccccagct tgaacagccc tatgtgttca ttaaacgttc tgatgcctta 480

agcaccaatc acggccacaa ggtcgttgag ctggttgcag aaatggacgg cattcagtac 540

ggtcgtagcg gtataacact gggagtactc gtgccacatg tgggcgaaac cccaattgca 600

taccgcaatg ttcttcttcg taagaacggt aataagggag ccggtggtca tagctatggc 660

atcgatctaa agtcttatga cttaggtgac gagcttggca ctgatcccat tgaagattat 720

gaacaaaact ggaacactaa gcatggcagt ggtgcactcc gtgaactcac tcgtgagctc 780

aatggaggtg cagtcactcg ctatgtcgac aacaatttct gtggcccaga tgggtaccct 840

cttgattgca tcaaagattt tctcgcacgc gcgggcaagt caatgtgcac tctttccgaa 900

caacttgatt acatcgagtc gaagagaggt gtctactgct gccgtgacca tgagcatgaa 960

attgcctggt tcactgagcg ctctgataag agctacgagc accagacacc cttcgaaatt 1020

aagagtgcca agaaatttga cactttcaaa ggggaatgcc caaagtttgt gtttcctctt 1080

aactcaaaag tcaaagtcat tcaaccacgt gttgaaaaga aaaagactga gggtttcatg 1140

gggcgtatac gctctgtgta ccctgttgca tctccacagg agtgtaacaa tatgcacttg 1200

tctaccttga tgaaatgtaa tcattgcgat gaagtttcat ggcagacgtg cgactttctg 1260

aaagccactt gtgaacattg tggcactgaa aatttagtta ttgaaggacc tactacatgt 1320

gggtacctac ctactaatgc tgtagtgaaa atgccatgtc ctgcctgtca agacccagag 1380

attggacctg agcatagtgt tgcagattat cacaaccact caaacattga aactcgactc 1440

cgcaagggag gtaggactag atgttttgga ggctgtgtgt ttgcctatgt tggctgctat 1500

aataagcgtg cctactgggt tcctcgtgct agtgctgata ttggctcagg ccatactggc 1560

attactggtg acaatgtgga gaccttgaat gaggatctcc ttgagatact gagtcgtgaa 1620

cgtgttaaca ttaacattgt tggcgatttt catttgaatg aagaggttgc catcattttg 1680

gcatctttct ctgcttctac aagtgccttt attgacacta taaagagtct tgattacaag 1740

tctttcaaaa ccattgttga gtcctgcggt aactataaag ttaccaaggg aaagcccgta 1800

aaaggtgctt ggaacattgg acaacagaga tcagttttaa caccactgtg tggttttccc 1860

tcacaggctg ctggtgttat cagatcaatt tttgcgcgca cacttgatgc agcaaaccac 1920

tcaattcctg atttgcaaag agcagctgtc accatacttg atggtatttc tgaacagtca 1980

ttacgtcttg tcgacgccat ggtttatact tcagacctgc tcaccaacag tgtcattatt 2040

atggcatatg taactggtgg tcttgtacaa cagacttctc agtggttgtc taatcttttg 2100

ggcactactg ttgaaaaact caggcctatc tttgaatgga ttgaggcgaa acttagtgca 2160

ggagttgaat ttctcaagga tgcttgggag attctcaaat ttctcattac aggtgttttt 2220

gacatcgtca agggtcaaat acaggttgct tcagataaca tcaaggattg tgtaaaatgc 2280

ttcattgatg ttgttaacaa ggcactcgaa atgtgcattg atcaagtcac tatcgctggc 2340

gcaaagttgc gatcactcaa cttaggtgaa gtcttcatcg ctcaaagcaa gggactttac 2400

cgtcagtgta tacgtggcaa ggagcagctg caactactca tgcctcttaa ggcaccaaaa 2460

gaagtaacct ttcttgaagg tgattcacat gacacagtac ttacctctga ggaggttgtt 2520

ctcaagaacg gtgaactcga agcactcgag acgcccgttg atagcttcac aaatggagct 2580

atcgttggca caccagtctg tgtaaatggc ctcatgctct tagagattaa ggacaaagaa 2640

caatactgcg cattgtctcc tggtttactg gctacaaaca atgtctttcg cttaaaaggg 2700

ggtgcaccaa ttaaaggtgt aacctttgga gaagatactg tttgggaagt tcaaggttac 2760

aagaatgtga gaatcacatt tgagcttgat gaacgtgttg acaaagtgct taatgaaaag 2820

tgctctgtct acactgttga atccggtacc gaagttactg agtttgcatg tgttgtagca 2880

gaggctgttg tgaagacttt acaaccagtt tctgatctcc ttaccaacat gggtattgat 2940

cttgatgagt ggagtgtagc tacattctac ttatttgatg atgctggtga agaaaacttt 3000

tcatcacgta tgtattgttc cttttaccct ccagatgagg aagaagagga cgatgcagag 3060

tgtgaggaag aagaaattga tgaaacctgt gaacatgagt acggtacaga ggatgattat 3120

caaggtctcc ctctggaatt tggtgcctca gctgaaacag ttcgagttga ggaagaagaa 3180

gaggaagact ggctggatga tactactgag caatcagaga ttgagccaga accagaacct 3240

acacctgaag aaccagttaa tcagtttact ggttatttaa aacttactga caatgttgcc 3300

attaaatgtg ttgacatcgt taaggaggca caaagtgcta atcctatggt gattgtaaat 3360

gctgctaaca tacacctgaa acatggtggt ggtgtagcag gtgcactcaa caaggcaacc 3420

aatggtgcca tgcaaaagga gagtgatgat tacattaagc taaatggccc tcttacagta 3480

ggagggtctt gtttgctttc tggacataat cttgctaaga agtgtctgca tgttgttgga 3540

cctaacctaa atgcaggtga ggacatccag cttcttaagg cagcatatga aaatttcaat 3600

tcacaggaca tcttacttgc accattgttg tcagcaggca tatttggtgc taaaccactt 3660

cagtctttac aagtgtgcgt gcagacggtt cgtacacagg tttatattgc agtcaatgac 3720

aaagctcttt atgagcaggt tgtcatggat tatcttgata acctgaagcc tagagtggaa 3780

gcacctaaac aagaggagcc accaaacaca gaagattcca aaactgagga gaaatctgtc 3840

gtacagaagc ctgtcgatgt gaagccaaaa attaaggcct gcattgatga ggttaccaca 3900

acactggaag aaactaagtt tcttaccaat aagttactct tgtttgctga tatcaatggt 3960

aagctttacc atgattctca gaacatgctt agaggtgaag atatgtcttt ccttgagaag 4020

gatgcacctt acatggtagg tgatgttatc actagtggtg atatcacttg tgttgtaata 4080

ccctccaaaa aggctggtgg cactactgag atgctctcaa gagctttgaa gaaagtgcca 4140

gttgatgagt atataaccac gtaccctgga caaggatgtg ctggttatac acttgaggaa 4200

gctaagactg ctcttaagaa atgcaaatct gcattttatg tactaccttc agaagcacct 4260

aatgctaagg aagagattct aggaactgta tcctggaatt tgagagaaat gcttgctcat 4320

gctgaagaga caagaaaatt aatgcctata tgcatggatg ttagagccat aatggcaacc 4380

atccaacgta agtataaagg aattaaaatt caagagggca tcgttgacta tggtgtccga 4440

ttcttctttt atactagtaa agagcctgta gcttctatta ttacgaagct gaactctcta 4500

aatgagccgc ttgtcacaat gccaattggt tatgtgacac atggttttaa tcttgaagag 4560

gctgcgcgct gtatgcgttc tcttaaagct cctgccgtag tgtcagtatc atcaccagat 4620

gctgttacta catataatgg atacctcact tcgtcatcaa agacatctga ggagcacttt 4680

gtagaaacag tttctttggc tggctcttac agagattggt cctattcagg acagcgtaca 4740

gagttaggtg ttgaatttct taagcgtggt gacaaaattg tgtaccacac tctggagagc 4800

cccgtcgagt ttcatcttga cggtgaggtt ctttcacttg acaaactaaa gagtctctta 4860

tccctgcggg aggttaagac tataaaagtg ttcacaactg tggacaacac taatctccac 4920

acacagcttg tggatatgtc tatgacatat ggacagcagt ttggtccaac atacttggat 4980

ggtgctgatg ttacaaaaat taaacctcat gtaaatcatg agggtaagac tttctttgta 5040

ctacctagtg atgacacact acgtagtgaa gctttcgagt actaccatac tcttgatgag 5100

agttttcttg gtaggtacat gtctgcttta aaccacacaa agaaatggaa atttcctcaa 5160

gttggtggtt taacttcaat taaatgggct gataacaatt gttatttgtc tagtgtttta 5220

ttagcacttc aacagcttga agtcaaattc aatgcaccag cacttcaaga ggcttattat 5280

agagcccgtg ctggtgatgc tgctaacttt tgtgcactca tactcgctta cagtaataaa 5340

actgttggcg agcttggtga tgtcagagaa actatgaccc atcttctaca gcatgctaat 5400

ttggaatctg caaagcgagt tcttaatgtg gtgtgtaaac attgtggtca gaaaactact 5460

accttaacgg gtgtagaagc tgtgatgtat atgggtactc tatcttatga taatcttaag 5520

acaggtgttt ccattccatg tgtgtgtggt cgtgatgcta cacaatatct agtacaacaa 5580

gagtcttctt ttgttatgat gtctgcacca cctgctgagt ataaattaca gcaaggtaca 5640

ttcttatgtg cgaatgagta cactggtaac tatcagtgtg gtcattacac tcatataact 5700

gctaaggaga ccctctatcg tattgacgga gctcacctta caaagatgtc agagtacaaa 5760

ggaccagtga ctgatgtttt ctacaaggaa acatcttaca ctacaaccat caagcctgtg 5820

tcgtataaac tcgatggagt tacttacaca gagattgaac caaaattgga tgggtattat 5880

aaaaaggata atgcttacta tacagagcag cctatagacc ttgtaccaac tcaaccatta 5940

ccaaatgcga gttttgataa tttcaaactc acatgttcta acacaaaatt tgctgatgat 6000

ttaaatcaaa tgacaggctt cacaaagcca gcttcacgag agctatctgt cacattcttc 6060

ccagacttga atggcgatgt agtggctatt gactatagac actattcagc gagtttcaag 6120

aaaggtgcta aattactgca taagccaatt gtttggcaca ttaaccaggc tacaaccaag 6180

acaacgttca aaccaaacac ttggtgttta cgttgtcttt ggagtacaaa gccagtagat 6240

acttcaaatt catttgaagt tctggcagta gaagacacac aaggaatgga caatcttgct 6300

tgtgaaagtc aacaacccac ctctgaagaa gtagtggaaa atcctaccat acagaaggaa 6360

gtcatagagt gtgacgtgaa aactaccgaa gttgtaggca atgtcatact taaaccatca 6420

gatgaaggtg ttaaagtaac acaagagtta ggtcatgagg atcttatggc tgcttatgtg 6480

gaaaacacaa gcattaccat taagaaacct aatgagcttt cactagcctt aggtttaaaa 6540

acaattgcca ctcatggtat tgctgcaatt aatagtgttc cttggagtaa aattttggct 6600

tatgtcaaac cattcttagg acaagcagca attacaacat caaattgcgc taagagatta 6660

gcacaacgtg tgtttaacaa ttatatgcct tatgtgttta cattattgtt ccaattgtgt 6720

acttttacta aaagtaccaa ttctagaatt agagcttcac tacctacaac tattgctaaa 6780

aatagtgtta agagtgttgc taaattatgt ttggatgccg gcattaatta tgtgaagtca 6840

cccaaatttt ctaaattgtt cacaatcgct atgtggctat tgttgttaag tatttgctta 6900

ggttctctaa tctgtgtaac tgctgctttt ggtgtactct tatctaattt tggtgctcct 6960

tcttattgta atggcgttag agaattgtat cttaattcgt ctaacgttac tactatggat 7020

ttctgtgaag gttcttttcc ttgcagcatt tgtttaagtg gattagactc ccttgattct 7080

tatccagctc ttgaaaccat tcaggtgacg atttcatcgt acaagctaga cttgacaatt 7140

ttaggtctgg ccgctgagtg ggttttggca tatatgttgt tcacaaaatt cttttattta 7200

ttaggtcttt cagctataat gcaggtgttc tttggctatt ttgctagtca tttcatcagc 7260

aattcttggc tcatgtggtt tatcattagt attgtacaaa tggcacccgt ttctgcaatg 7320

gttaggatgt acatcttctt tgcttctttc tactacatat ggaagagcta tgttcatatc 7380

atggatggtt gcacctcttc gacttgcatg atgtgctata agcgcaatcg tgccacacgc 7440

gttgagtgta caactattgt taatggcatg aagagatctt tctatgtcta tgcaaatgga 7500

ggccgtggct tctgcaagac tcacaattgg aattgtctca attgtgacac attttgcact 7560

ggtagtacat tcattagtga tgaagttgct cgtgatttgt cactccagtt taaaagacca 7620

atcaacccta ctgaccagtc atcgtatatt gttgatagtg ttgctgtgaa aaatggcgcg 7680

cttcacctct actttgacaa ggctggtcaa aagacctatg agagacatcc tctctcccat 7740

tttgtcaatt tagacaattt gagagctaac aacactaaag gttcactgcc tattaatgtc 7800

atagtttttg atggcaagtc caaatgcgac gagtctgctt ctaagtctgc ttctgtgtac 7860

tacagtcagc tgatgtgcca acctattctg ttgcttgacc aagctcttgt atcagacgtt 7920

ggagatagta ctgaagtttc cgttaagatg tttgatgctt atgtcgacac cttttcagca 7980

acttttagtg ttcctatgga aaaacttaag gcacttgttg ctacagctca cagcgagtta 8040

gcaaagggtg tagctttaga tggtgtcctt tctacattcg tgtcagctgc ccgacaaggt 8100

gttgttgata ccgatgttga cacaaaggat gttattgaat gtctcaaact ttcacatcac 8160

tctgacttag aagtgacagg tgacagttgt aacaatttca tgctcaccta taataaggtt 8220

gaaaacatga cgcccagaga tcttggcgca tgtattgact gtaatgcaag gcatatcaat 8280

gcccaagtag caaaaagtca caatgtttca ctcatctgga atgtaaaaga ctacatgtct 8340

ttatctgaac agctgcgtaa acaaattcgt agtgctgcca agaagaacaa catacctttt 8400

agactaactt gtgctacaac tagacaggtt gtcaatgtca taactactaa aatctcactc 8460

aagggtggta agattgttag tacttgtttt aaacttatgc ttaaggccac attattgtgc 8520

gttcttgctg cattggtttg ttatatcgtt atgccagtac atacattgtc aatccatgat 8580

ggttacacaa atgaaatcat tggttacaaa gccattcagg atggtgtcac tcgtgacatc 8640

atttctactg atgattgttt tgcaaataaa catgctggtt ttgacgcatg gtttagccag 8700

cgtggtggtt catacaaaaa tgacaaaagc tgccctgtag tagctgctat cattacaaga 8760

gagattggtt tcatagtgcc tggcttaccg ggtactgtgc tgagagcaat caatggtgac 8820

ttcttgcatt ttctacctcg tgtttttagt gctgttggca acatttgcta cacaccttcc 8880

aaactcattg agtatagtga ttttgctacc tctgcttgcg ttcttgctgc tgagtgtaca 8940

atttttaagg atgctatggg caaacctgtg ccatattgtt atgacactaa tttgctagag 9000

ggttctattt cttatagtga gcttcgtcca gacactcgtt atgtgcttat ggatggttcc 9060

atcatacagt ttcctaacac ttacctggag ggttctgtta gagtagtaac aacttttgat 9120

gctgagtact gtagacatgg tacatgcgaa aggtcagaag taggtatttg cctatctacc 9180

agtggtagat gggttcttaa taatgagcat tacagagctc tatcaggagt tttctgtggt 9240

gttgatgcga tgaatctcat agctaacatc tttactcctc ttgtgcaacc tgtgggtgct 9300

ttagatgtgt ctgcttcagt agtggctggt ggtattattg ccatattggt gacttgtgct 9360

gcctactact ttatgaaatt cagacgtgct tttggtgagt acaaccatgt tgttgctgct 9420

aatgcacttt tgtttttgat gtctttcact atactctgtc tggcaccagc ttacagcttt 9480

ctgccgggag tctactcagt cttttacttg tacttgacat tctatttcac caatgatgtt 9540

tcattcttgg ctcaccttca atggtttgcc atgttttctc ctattgtgcc tttttggata 9600

acagcaatct atgtattctg tatttctctg aagcactgcc attggttctt taacaactat 9660

cttaggaaaa gagtcatgtt taatggagtt acatttagta ccttcgagga ggctgctttg 9720

tgtacctttt tgctcaacaa ggaaatgtac ctaaaattgc gtagcgagac actgttgcca 9780

cttacacagt ataacaggta tcttgctcta tataacaagt acaagtattt cagtggagcc 9840

ttagatacta ccagctatcg tgaagcagct tgctgccact tagcaaaggc tctaaatgac 9900

tttagcaact caggtgctga tgttctctac caaccaccac agacatcaat cacttctgct 9960

gttctgcaga gtggttttag gaaaatggca ttcccgtcag gcaaagttga agggtgcatg 10020

gtacaagtaa cctgtggaac tacaactctt aatggattgt ggttggatga cacagtatac 10080

tgtccaagac atgtcatttg cacagcagaa gacatgctta atcctaacta tgaagatctg 10140

ctcattcgca aatccaacca tagctttctt gttcaggctg gcaatgttca acttcgtgtt 10200

attggccatt ctatgcaaaa ttgtctgctt aggcttaaag ttgatacttc taaccctaag 10260

acacccaagt ataaatttgt ccgtatccaa cctggtcaaa cattttcagt tctagcatgc 10320

tacaatggtt caccatctgg tgtttatcag tgtgccatga gacctaatca taccattaaa 10380

ggttctttcc ttaatggatc atgtggtagt gttggtttta acattgatta tgattgcgtg 10440

tctttctgct atatgcatca tatggagctt ccaacaggag tacacgctgg tactgactta 10500

gaaggtaaat tctatggtcc atttgttgac agacaaactg cacaggctgc aggtacagac 10560

acaaccataa cattaaatgt tttggcatgg ctgtatgctg ctgttatcaa tggtgatagg 10620

tggtttctta atagattcac cactactttg aatgacttta accttgtggc aatgaagtac 10680

aactatgaac ctttgacaca agatcatgtt gacatattgg gacctctttc tgctcaaaca 10740

ggaattgccg tcttagatat gtgtgctgct ttgaaagagc tgctgcagaa tggtatgaat 10800

ggtcgtacta tccttggtag cactatttta gaagatgagt ttacaccatt tgatgttgtt 10860

agacaatgct ctggtgttac cttccaaggt aagttcaaga aaattgttaa gggcactcat 10920

cattggatgc ttttaacttt cttgacatca ctattgattc ttgttcaaag tacacagtgg 10980

tcactgtttt tctttgttta cgagaatgct ttcttgccat ttactcttgg tattatggca 11040

attgctgcat gtgctatgct gcttgttaag cataagcacg cattcttgtg cttgtttctg 11100

ttaccttctc ttgcaacagt tgcttacttt aatatggtct acatgcctgc tagctgggtg 11160

atgcgtatca tgacatggct tgaattggct gacactagct tgtctggtta taggcttaag 11220

gattgtgtta tgtatgcttc agctttagtt ttgcttattc tcatgacagc tcgcactgtt 11280

tatgatgatg ctgctagacg tgtttggaca ctgatgaatg tcattacact tgtttacaaa 11340

gtctactatg gtaatgcttt agatcaagct atttccatgt gggccttagt tatttctgta 11400

acctctaact attctggtgt cgttacgact atcatgtttt tagctagagc tatagtgttt 11460

gtgtgtgttg agtattaccc attgttattt attactggca acaccttaca gtgtatcatg 11520

cttgtttatt gtttcttagg ctattgttgc tgctgctact ttggcctttt ctgtttactc 11580

aaccgttact tcaggcttac tcttggtgtt tatgactact tggtctctac acaagaattt 11640

aggtatatga actcccaggg gcttttgcct cctaagagta gtattgatgc tttcaagctt 11700

aacattaagt tgttgggtat tggaggtaaa ccatgtatca aggttgctac tgtacagtct 11760

aaaatgtctg acgtaaagtg cacatctgtg gtactgctct cggttcttca acaacttaga 11820

gtagagtcat cttctaaatt gtgggcacaa tgtgtacaac tccacaatga tattcttctt 11880

gcaaaagaca caactgaagc tttcgagaag atggtttctc ttttgtctgt tttgctatcc 11940

atgcagggtg ctgtagacat taataggttg tgcgaggaaa tgctcgataa ccgtgctact 12000

cttcaggcta ttgcttcaga atttagttct ttaccatcat atgccgctta tgccactgcc 12060

caggaggcct atgagcaggc tgtagctaat ggtgattctg aagtcgttct caaaaagtta 12120

aagaaatctt tgaatgtggc taaatctgag tttgaccgtg atgctgccat gcaacgcaag 12180

ttggaaaaga tggcagatca ggctatgacc caaatgtaca aacaggcaag atctgaggac 12240

aagagggcaa aagtaactag tgctatgcaa acaatgctct tcactatgct taggaagctt 12300

gataatgatg cacttaacaa cattatcaac aatgcgcgtg atggttgtgt tccactcaac 12360

atcataccat tgactacagc agccaaactc atggttgttg tccctgatta tggtacctac 12420

aagaacactt gtgatggtaa cacctttaca tatgcatctg cactctggga aatccagcaa 12480

gttgttgatg cggatagcaa gattgttcaa cttagtgaaa ttaacatgga caattcacca 12540

aatttggctt ggcctcttat tgttacagct ctaagagcca actcagctgt taaactacag 12600

aataatgaac tgagtccagt agcactacga cagatgtcct gtgcggctgg taccacacaa 12660

acagcttgta ctgatgacaa tgcacttgcc tactataaca attcgaaggg aggtaggttt 12720

gtgctggcat tactatcaga ccaccaagat ctcaaatggg ctagattccc taagagtgat 12780

ggtacaggta caatttacac agaactggaa ccaccttgta ggtttgttac agacacacca 12840

aaagggccta aagtgaaata cttgtacttc atcaaaggct taaacaacct aaatagaggt 12900

atggtgctgg gcagtttagc tgctacagta cgtcttcagg ctggaaatgc tacagaagta 12960

cctgccaatt caactgtgct ttccttctgt gcttttgcag tagaccctgc taaagcatat 13020

aaggattacc tagcaagtgg aggacaacca atcaccaact gtgtgaagat gttgtgtaca 13080

cacactggta caggacaggc aattactgta acaccagaag ctaacatgga ccaagagtcc 13140

tttggtggtg cttcatgttg tctgtattgt agatgccaca ttgaccatcc aaatcctaaa 13200

ggattctgtg acttgaaagg taagtacgtc caaataccta ccacttgtgc taatgaccca 13260

gtgggtttta cacttagaaa cacagtctgt accgtctgcg gaatgtggaa aggttatggc 13320

tgtagttgtg accaactccg cgaacccttg atgcagtctg cggatgcatc aacgttttta 13380

aacgggtttg cggtgtaagt gcagcccgtc ttacaccgtg cggcacaggc actagtactg 13440

atgtcgtcta cagggctttt gatatttaca acgaaaaagt tgctggtttt gcaaagttcc 13500

taaaaactaa ttgctgtcgc ttccaggaga aggatgagga aggcaattta ttagactctt 13560

actttgtagt taagaggcat actatgtcta actaccaaca tgaagagact atttataact 13620

tggttaaaga ttgtccagcg gttgctgtcc atgacttttt caagtttaga gtagatggtg 13680

acatggtacc acatatatca cgtcagcgtc taactaaata cacaatggct gatttagtct 13740

atgctctacg tcattttgat gagggtaatt gtgatacatt aaaagaaata ctcgtcacat 13800

acaattgctg tgatgatgat tatttcaata agaaggattg gtatgacttc gtagagaatc 13860

ctgacatctt acgcgtatat gctaacttag gtgagcgtgt acgccaatca ttattaaaga 13920

ctgtacaatt ctgcgatgct atgcgtgatg caggcattgt aggcgtactg acattagata 13980

atcaggatct taatgggaac tggtacgatt tcggtgattt cgtacaagta gcaccaggct 14040

gcggagttcc tattgtggat tcatattact cattgctgat gcccatcctc actttgacta 14100

gggcattggc tgctgagtcc catatggatg ctgatctcgc aaaaccactt attaagtggg 14160

atttgctgaa atatgatttt acggaagaga gactttgtct cttcgaccgt tattttaaat 14220

attgggacca gacataccat cccaattgta ttaactgttt ggatgatagg tgtatccttc 14280

attgtgcaaa ctttaatgtg ttattttcta ctgtgtttcc acctacaagt tttggaccac 14340

tagtaagaaa aatatttgta gatggtgttc cttttgttgt ttcaactgga taccattttc 14400

gtgagttagg agtcgtacat aatcaggatg taaacttaca tagctcgcgt ctcagtttca 14460

aggaactttt agtgtatgct gctgatccag ctatgcatgc agcttctggc aatttattgc 14520

tagataaacg cactacatgc ttttcagtag ctgcactaac aaacaatgtt gcttttcaaa 14580

ctgtcaaacc cggtaatttt aataaagact tttatgactt tgctgtgtct aaaggtttct 14640

ttaaggaagg aagttctgtt gaactaaaac acttcttctt tgctcaggat ggcaacgctg 14700

ctatcagtga ttatgactat tatcgttata atctgccaac aatgtgtgat atcagacaac 14760

tcctattcgt agttgaagtt gttgataaat actttgattg ttacgatggt ggctgtatta 14820

atgccaacca agtaatcgtt aacaatctgg ataaatcagc tggtttccca tttaataaat 14880

ggggtaaggc tagactttat tatgactcaa tgagttatga ggatcaagat gcacttttcg 14940

cgtatactaa gcgtaatgtc atccctacta taactcaaat gaatcttaag tatgccatta 15000

gtgcaaagaa tagagctcgc accgtagctg gtgtctctat ctgtagtact atgacaaata 15060

gacagtttca tcagaaatta ttgaagtcaa tagccgccac tagaggagct actgtggtaa 15120

ttggaacaag caagttttac ggtggctggc ataatatgtt aaaaactgtt tacagtgatg 15180

tagaaactcc acaccttatg ggttgggatt atccaaaatg tgacagagcc atgcctaaca 15240

tgcttaggat aatggcctct cttgttcttg ctcgcaaaca taacacttgc tgtaacttat 15300

cacaccgttt ctacaggtta gctaacgagt gtgcgcaagt attaagtgag atggtcatgt 15360

gtggcggctc actatatgtt aaaccaggtg gaacatcatc cggtgatgct acaactgctt 15420

atgctaatag tgtctttaac atttgtcaag ctgttacagc caatgtaaat gcacttcttt 15480

caactgatgg taataagata gctgacaagt atgtccgcaa tctacaacac aggctctatg 15540

agtgtctcta tagaaatagg gatgttgatc atgaattcgt ggatgagttt tacgcttacc 15600

tgcgtaaaca tttctccatg atgattcttt ctgatgatgc cgttgtgtgc tataacagta 15660

actatgcggc tcaaggttta gtagctagca ttaagaactt taaggcagtt ctttattatc 15720

aaaataatgt gttcatgtct gaggcaaaat gttggactga gactgacctt actaaaggac 15780

ctcacgaatt ttgctcacag catacaatgc tagttaaaca aggagatgat tacgtgtacc 15840

tgccttaccc agatccatca agaatattag gcgcaggctg ttttgtcgat gatattgtca 15900

aaacagatgg tacacttatg attgaaaggt tcgtgtcact ggctattgat gcttacccac 15960

ttacaaaaca tcctaatcag gagtatgctg atgtctttca cttgtattta caatacatta 16020

gaaagttaca tgatgagctt actggccaca tgttggacat gtattccgta atgctaacta 16080

atgataacac ctcacggtac tgggaacctg agttttatga ggctatgtac acaccacata 16140

cagtcttgca ggctgtaggt gcttgtgtat tgtgcaattc acagacttca cttcgttgcg 16200

gtgcctgtat taggagacca ttcctatgtt gcaagtgctg ctatgaccat gtcatttcaa 16260

catcacacaa attagtgttg tctgttaatc cctatgtttg caatgcccca ggttgtgatg 16320

tcactgatgt gacacaactg tatctaggag gtatgagcta ttattgcaag tcacataagc 16380

ctcccattag ttttccatta tgtgctaatg gtcaggtttt tggtttatac aaaaacacat 16440

gtgtaggcag tgacaatgtc actgacttca atgcgatagc aacatgtgat tggactaatg 16500

ctggcgatta catacttgcc aacacttgta ctgagagact caagcttttc gcagcagaaa 16560

cgctcaaagc cactgaggaa acatttaagc tgtcatatgg tattgccact gtacgcgaag 16620

tactctctga cagagaattg catctttcat gggaggttgg aaaacctaga ccaccattga 16680

acagaaacta tgtctttact ggttaccgtg taactaaaaa tagtaaagta cagattggag 16740

agtacacctt tgaaaaaggt gactatggtg atgctgttgt gtacagaggt actacgacat 16800

acaagttgaa tgttggtgat tactttgtgt tgacatctca cactgtaatg ccacttagtg 16860

cacctactct agtgccacaa gagcactatg tgagaattac tggcttgtac ccaacactca 16920

acatctcaga tgagttttct agcaatgttg caaattatca aaaggtcggc atgcaaaagt 16980

actctacact ccaaggacca cctggtactg gtaagagtca ttttgccatc ggacttgctc 17040

tctattaccc atctgctcgc atagtgtata cggcatgctc tcatgcagct gttgatgccc 17100

tatgtgaaaa ggcattaaaa tatttgccca tagataaatg tagtagaatc atacctgcgc 17160

gtgcgcgcgt agagtgtttt gataaattca aagtgaattc aacactagaa cagtatgttt 17220

tctgcactgt aaatgcattg ccagaaacaa ctgctgacat tgtagtcttt gatgaaatct 17280

ctatggctac taattatgac ttgagtgttg tcaatgctag acttcgtgca aaacactacg 17340

tctatattgg cgatcctgct caattaccag ccccccgcac attgctgact aaaggcacac 17400

tagaaccaga atattttaat tcagtgtgca gacttatgaa aacaataggt ccagacatgt 17460

tccttggaac ttgtcgccgt tgtcctgctg aaattgttga cactgtgagt gctttagttt 17520

atgacaataa gctaaaagca cacaaggaga agtcagctca atgcttcaaa atgttctaca 17580

aaggtgttat tacacatgat gtttcatctg caatcaacag acctcaaata ggcgttgtaa 17640

gagaatttct tacacgcaat cctgcttgga gaaaagctgt ttttatctca ccttataatt 17700

cacagaacgc tgtagcttca aaaatcttag gattgcctac gcagactgtt gattcatcac 17760

agggttctga atatgactat gtcatattca cacaaactac tgaaacagca cactcttgta 17820

atgtcaaccg tttcaatgtg gctatcacaa gggcaaaaat tggcattttg tgcataatgt 17880

ctgatagaga tctttatgac aaactgcaat ttacaagtct agaaatacca cgtcgcaatg 17940

tggctacatt acaagcagaa aatgtaactg gactttttaa ggactgtagt aagatcatta 18000

ctggtcttca tcctacacag gcacctacac acctcagcgt tgatataaag ttcaagactg 18060

aaggattatg tgttgacata ccaggcatac caaaggacat gacctaccgt agactcatct 18120

ctatgatggg tttcaaaatg aattaccaag tcaatggtta ccctaatatg tttatcaccc 18180

gcgaagaagc tattcgtcac gttcgtgcgt ggattggctt tgatgtagag ggctgtcatg 18240

caactagaga tgctgtgggt actaacctac ctctccagct aggattttct acaggtgtta 18300

acttagtagc tgtaccgact ggttatgttg acactgaaaa taacacagaa ttcaccagag 18360

ttaatgcaaa acctccacca ggtgaccagt ttaaacatct tataccactc atgtataaag 18420

gcttgccctg gaatgtagtg cgtattaaga tagtacaaat gctcagtgat acactgaaag 18480

gattgtcaga cagagtcgtg ttcgtccttt gggcgcatgg ctttgagctt acatcaatga 18540

agtactttgt caagattgga cctgaaagaa cgtgttgtct gtgtgacaaa cgtgcaactt 18600

gcttttctac ttcatcagat acttatgcct gctggaatca ttctgtgggt tttgactatg 18660

tctataaccc atttatgatt gatgttcagc agtggggctt tacgggtaac cttcagagta 18720

accatgacca acattgccag gtacatggaa atgcacatgt ggctagttgt gatgctatca 18780

tgactagatg tttagcagtc catgagtgct ttgttaagcg cgttgattgg tctgttgaat 18840

accctattat aggagatgaa ctgagggtta attctgcttg cagaaaagta caacacatgg 18900

ttgtgaagtc tgcattgctt gctgataagt ttccagttct tcatgacatt ggaaatccaa 18960

aggctatcaa gtgtgtgcct caggctgaag tagaatggaa gttctacgat gctcagccat 19020

gtagtgacaa agcttacaaa atagaggagc tcttctattc ttatgctaca catcacgata 19080

aattcactga tggtgtttgt ttgttttgga attgtaacgt tgatcgttac ccagccaatg 19140

caattgtgtg taggtttgac acaagagtct tgtcaaactt gaacttacca ggctgtgatg 19200

gtggtagttt gtatgtgaat aagcatgcat tccacactcc agctttcgat aaaagtgcat 19260

ttactaattt aaagcaattg cctttctttt actattctga tagtccttgt gagtctcatg 19320

gcaaacaagt agtgtcggat attgattatg ttccactcaa atctgctacg tgtattacac 19380

gatgcaattt aggtggtgct gtttgcagac accatgcaaa tgagtaccga cagtacttgg 19440

atgcatataa tatgatgatt tctgctggat ttagcctatg gatttacaaa caatttgata 19500

cttataacct gtggaataca tttaccaggt tacagagttt agaaaatgtg gcttataatg 19560

ttgttaataa aggacacttt gatggacacg ccggcgaagc acctgtttcc atcattaata 19620

atgctgttta cacaaaggta gatggtattg atgtggagat ctttgaaaat aagacaacac 19680

ttcctgttaa tgttgcattt gagctttggg ctaagcgtaa cattaaacca gtgccagaga 19740

ttaagatact caataatttg ggtgttgata tcgctgctaa tactgtaatc tgggactaca 19800

aaagagaagc cccagcacat gtatctacaa taggtgtctg cacaatgact gacattgcca 19860

agaaacctac tgagagtgct tgttcttcac ttactgtctt gtttgatggt agagtggaag 19920

gacaggtaga cctttttaga aacgcccgta atggtgtttt aataacagaa ggttcagtca 19980

aaggtctaac accttcaaag ggaccagcac aagctagcgt caatggagtc acattaattg 20040

gagaatcagt aaaaacacag tttaactact ttaagaaagt agacggcatt attcaacagt 20100

tgcctgaaac ctactttact cagagcagag acttagagga ttttaagccc agatcacaaa 20160

tggaaactga ctttctcgag ctcgctatgg atgaattcat acagcgatat aagctcgagg 20220

gctatgcctt cgaacacatc gtttatggag atttcagtca tggacaactt ggcggtcttc 20280

atttaatgat aggcttagcc aagcgctcac aagattcacc acttaaatta gaggatttta 20340

tccctatgga cagcacagtg aaaaattact tcataacaga tgcgcaaaca ggttcatcaa 20400

aatgtgtgtg ttctgtgatt gatcttttac ttgatgactt tgtcgagata ataaagtcac 20460

aagatttgtc agtgatttca aaagtggtca aggttacaat tgactatgct gaaatttcat 20520

tcatgctttg gtgtaaggat ggacatgttg aaaccttcta cccaaaacta caagcaagtc 20580

aagcgtggca accaggtgtt gcgatgccta acttgtacaa gatgcaaaga atgcttcttg 20640

aaaagtgtga ccttcagaat tatggtgaaa atgctgttat accaaaagga ataatgatga 20700

atgtcgcaaa gtatactcaa ctgtgtcaat acttaaatac acttacttta gctgtaccct 20760

acaacatgag agttattcac tttggtgctg gctctgataa aggagttgca ccaggtacag 20820

ctgtgctcag acaatggttg ccaactggca cactacttgt cgattcagat cttaatgact 20880

tcgtctccga cgcagattct actttaattg gagactgtgc aacagtacat acggctaata 20940

aatgggacct tattattagc gatatgtatg accctaggac caaacatgtg acaaaagaga 21000

atgactctaa agaagggttt ttcacttatc tgtgtggatt tataaagcaa aaactagccc 21060

tgggtggttc tatagctgta aagataacag agcattcttg gaatgctgac ctttacaagc 21120

ttatgggcca tttctcatgg tggacagctt ttgttacaaa tgtaaatgca tcacatcgg 21180

aagcattttt aattggggct aactatcttg gcaagccgaa ggaacaaatt gatggctata 21240

ccatgcatgc taactacatt ttctggagga acacaaatcc tatccagttg tcttcctatt 21300

cactctttga catgagcaaa tttcctctta aattaagagg aactgctgta atgtctctta 21360

aggagaatca aatcaatgat atgatttatt ctcttctgga aaaaggtagg cttatcatta 21420

gagaaaacaa cagagttgtg gtttcaagtg atattcttgt taacaactaa acgaacatgt 21480

ttattttctt attatttctt actctcacta gtggtagtga ccttgaccgg tgcaccactt 21540

ttgatgatgt tcaagctcct aattacactc aacatacttc atctatgagg ggggtttact 21600

atcctgatga aatttttaga tcagacactc tttatttaac tcaggattta tttcttccat 21660

tttattctaa tgttacaggg tttcatacta ttaatcatac gtttgacaac cctgtcatac 21720

cttttaagga tggtatttat tttgctgcca cagagaaatc aaatgttgtc cgtggttggg 21780

tttttggttc taccatgaac aacaagtcac agtcggtgat tattattaac aattctacta 21840

atgttgttat acgagcatgt aactttgaat tgtgtgacaa ccctttcttt gctgtttcta 21900

aacccatggg tacacagaca catactatga tattcgataa tgcatttaat tgcactttcg 21960

agtacatatc tgatgccttt tcgcttgatg tttcagaaaa gtcaggtaat tttaaacact 22020

tacgagagtt tgtgtttaaa aataaagatg ggtttctcta tgtttataag ggctatcaac 22080

ctatagatgt agttcgtgat ctaccttctg gttttaacac tttgaaacct atttttaagt 22140

tgcctcttgg tattaacatt acaaatttta gagccattct tacagccttt tcacctgctc 22200

aagacacttg gggcacgtca gctgcagcct attttgttgg ctatttaaag ccaactacat 22260

ttatgctcaa gtatgatgaa aatggtacaa tcacagatgc tgttgattgt tctcaaaatc 22320

cacttgctga actcaaatgc tctgttaaga gctttgagat tgacaaagga atttaccaga 22380

cctctaattt cagggttgtt ccctcaggag atgttgtgag attccctaat attacaaact 22440

tgtgtccttt tggagaggtt tttaatgcta ctaaattccc ttctgtctat gcatgggaga 22500

gaaaaaaaat ttctaattgt gttgctgatt actctgtgct ctacaactca acattttttt 22560

caacctttaa gtgctatggc gtttctgcca ctaagttgaa tgatctttgc ttctccaatg 22620

tctatgcaga ttcttttgta gtcaagggag atgatgtaag acaaatagcg ccaggacaaa 22680

ctggtgttat tgctgattat aattataaat tgccagatga tttcatgggt tgtgtccttg 22740

cttggaatac taggaacatt gatgctactt caactggtaa ttataattat aaatataggt 22800

atcttagaca tggcaagctt aggccctttg agagagacat atctaatgtg cctttctccc 22860

ctgatggcaa accttgcacc ccacctgctc ttaattgtta ttggccatta aatgattatg 22920

gtttttacac cactactggc attggctacc aaccttacag agttgtagta ctttcttttg 22980

aacttttaaa tgcaccggcc acggtttgtg gaccaaaatt atccactgac cttattaaga 23040

accagtgtgt caattttaat tttaatggac tcactggtac tggtgtgtta actccttctt 23100

caaagagatt tcaaccattt caacaatttg gccgtgatgt ttctgatttc actgattccg 23160

ttcgagatcc taaaacatct gaaatattag acatttcacc ttgctctttt gggggtgtaa 23220

gtgtaattac acctggaaca aatgcttcat ctgaagttgc tgttctatat caagatgtta 23280

actgcactga tgtttctaca gcaattcatg cagatcaact cacaccagct tggcgcatat 23340

attctactgg aaacaatgta ttccagactc aagcaggctg tcttatagga gctgagcatg 23400

tcgacacttc ttatgagtgc gacattccta ttggagctgg catttgtgct agttaccata 23460

cagtttcttt attacgtagt actagccaaa aatctattgt ggcttatact atgtctttag 23520

gtgctgatag ttcaattgct tactctaata acaccattgc tatacctact aacttttcaa 23580

ttagcattac tacagaagta atgcctgttt ctatggctaa aacctccgta gattgtaata 23640

tgtacatctg cggagattct actgaatgtg ctaatttgct tctccaatat ggtagctttt 23700

gcacacaact aaatcgtgca ctctcaggta ttgctgctga acaggatcgc aacacacgtg 23760

aagtgttcgc tcaagtcaaa caaatgtaca aaaccccaac tttgaaatat tttggtggtt 23820

ttaatttttc acaaatatta cctgaccctc taaagccaac taagaggtct tttattgagg 23880

acttgctctt taataaggtg acactcgctg atgctggctt catgaagcaa tatggcgaat 23940

gcctaggtga tattaatgct agagatctca tttgtgcgca gaagttcaat ggacttacag 24000

tgttgccacc tctgctcact gatgatatga ttgctgccta cactgctgct ctagttagtg 24060

gtactgccac tgctggatgg acatttggtg ctggcgctgc tcttcaaata ccttttgcta 24120

tgcaaatggc atataggttc aatggcattg gagttaccca aaatgttctc tatgagaacc 24180

aaaaacaaat cgccaaccaa tttaacaagg cgattagtca aattcaagaa tcacttacaa 24240

caacatcaac tgcattgggc aagctgcaag acgttgttaa ccagaatgct caagcattaa 24300

acacacttgt taaacaactt agctctaatt ttggtgcaat ttcaagtgtg ctaaatgata 24360

tcctttcgcg acttgataaa gtcgaggcgg aggtacaaat tgacaggtta attacaggca 24420

gacttcaaag ccttcaaacc tatgtaacac aacaactaat cagggctgct gaaatcaggg 24480

cttctgctaa tcttgctgct actaaaatgt ctgagtgtgt tcttggacaa tcaaaaagag 24540

ttgacttttg tggaaagggc taccacctta tgtccttccc acaagcagcc ccgcatggtg 24600

ttgtcttcct acatgtcacg tatgtgccat cccaggagag gaacttcacc acagcgccag 24660

caatttgtca tgaaggcaaa gcatacttcc ctcgtgaagg tgtttttgtg tttaatggca 24720

cttcttggtt tattacacag aggaacttct tttctccaca aataattact acagacaata 24780

catttgtctc aggaaattgt gatgtcgtta ttggcatcat taacaacaca gtttatgatc 24840

ctctgcaacc tgagcttgac tcattcaaag aagagctgga caagtacttc aaaaatcata 24900

catcaccaga tgttgatctt ggcgacattt caggcattaa cgcttctgtc gtcaacattc 24960

aaaaagaaat tgaccgcctc aatgaggtcg ctaaaaattt aaatgaatca ctcattgacc 25020

ttcaagaatt gggaaaatat gagcaatata ttaaatggcc ttggtatgtt tggctcggct 25080

tcattgctgg actaattgcc atcgtcatgg ttacaatctt gctttgttgc atgactagtt 25140

gttgcagttg cctcaagggt gcatgctctt gtggttcttg ctgcaagttt gatgaggatg 25200

actctgagcc agttctcaag ggtgtcaaat tacattacac ataaacgaac ttatggattt 25260

gtttatgaga ttttttactc ttggatcaat tactgcacag ccagtaaaaa ttgacaatgc 25320

ttctcctgca agtactgttc atgctacagc aacgataccg ctacaagcct cactcccttt 25380

cggatggctt gttattggcg ttgcatttct tgctgttttt cagagcgcta ccaaaataat 25440

tgcgctcaat aaaagatggc agctagccct ttataagggc ttccagttca tttgcaattt 25500

actgctgcta tttgttacca tctattcaca tcttttgctt gtcgctgcag gtatggaggc 25560

gcaatttttg tacctctatg ccttgatata ttttctacaa tgcatcaacg catgtagaat 25620

tattatgaga tgttggcttt gttggaagtg caaatccaag aacccattac tttatgatgc 25680

caactacttt gtttgctggc acacacataa ctatgactac tgtataccat ataacagtgt 25740

cacagataca attgtcgtta ctgaaggtga cggcatttca acaccaaaac tcaaagaaga 25800

ctaccaaatt ggtggttatt ctgaggatag gcactcaggt gttaaagact atgtcgttgt 25860

acatggctat ttcaccgaag tttactacca gcttgagtct acacaaatta ctacagacac 25920

tggtattgaa aatgctacat tcttcatctt taacaagctt gttaaagacc caccgaatgt 25980

gcaaatacac acaatcgacg gctcttcagg agttgctaat ccagcaatgg atccaattta 26040

tgatgagccg acgacgacta ctagcgtgcc tttgtaagca caagaaagtg agtacgaact 26100

tatgtactca ttcgtttcgg aagaaacagg tacgttaata gttaatagcg tacttctttt 26160

tcttgctttc gtggtattct tgctagtcac actagccatc cttactgcgc ttcgattgtg 26220

tgcgtactgc tgcaatattg ttaacgtgag tttagtaaaa ccaacggttt acgtctactc 26280

gcgtgttaaa aatctgaact cttctgaagg agttcctgat cttctggtct aaacgaacta 26340

actattatta ttattctgtt tggaacttta acattgctta tcatggcaga caacggtact 26400

attaccgttg aggagcttaa acaactcctg gaacaatgga acctagtaat aggtttccta 26460

ttcctagcct ggattatgtt actacaattt gcctattcta atcggaacag gtttttgtac 26520

ataataaagc ttgttttcct ctggctcttg tggccagtaa cacttgcttg ttttgtgctt 26580

gctgctgtct acagaattaa ttgggtgact ggcgggattg cgattgcaat ggcttgtatt 26640

gtaggcttga tgtggcttag ctacttcgtt gcttccttca ggctgtttgc tcgtacccgc 26700

tcaatgtggt cattcaaccc agaaacaaac attcttctca atgtgcctct ccgggggaca 26760

attgtgacca gaccgctcat ggaaagtgaa cttgtcattg gtgctgtgat cattcgtggt 26820

cacttgcgaa tggccggaca ctccctaggg cgctgtgaca ttaaggacct gccaaaagag 26880

atcactgtgg ctacatcacg aacgctttct tattacaaat taggagcgtc gcagcgtgta 26940

ggcactgatt caggttttgc tgcatacaac cgctaccgta ttggaaacta taaattaaat 27000

acagaccacg ccggtagcaa cgacaatatt gctttgctag tacagtaagt gacaacagat 27060

gtttcatctt gttgacttcc aggttacaat agcagagata ttgattatca ttatgaggac 27120

tttcaggatt gctatttgga atcttgacgt tataataagt tcaatagtga gacaattatt 27180

taagcctcta actaagaaga attattcgga gttagatgat gaagaaccta tggagttaga 27240

ttatccataa aacgaacatg aaaattattc tcttcctgac attgattgta tttacatctt 27300

gcgagctata tcactatcag gagtgtgtta gaggtacgac tgtactacta aaagaacctt 27360

gcccatcagg aacatacgag ggcaattcac catttcaccc tcttgctgac aataaatttg 27420

cactaacttg cactagcaca cactttgctt ttgcttgtgc tgacggtact cgacatacct 27480

atcagctgcg tgcaagatca gtttcaccaa aacttttcat cagacaagag gaggttcaac 27540

aagagctcta ctcgccactt tttctcattg ttgctgctct agtattttta atactttgct 27600

tcaccattaa gagaaagaca gaatgaatga gctcacttta attgacttct atttgtgctt 27660

tttagccttt ctgctattcc ttgttttaat aatgcttatt atattttggt tttcactcga 27720

aatccaggat ctagaagaac cttgtaccaa agtctaaacg aacatgaaac ttctcattgt 27780

tttgacttgt atttctctat gcagttgcat acgcactgta gtacagcgct gtgcatctaa 27840

taaacctcat gtgcttgaag atccttgtaa ggtacaacac taggggtaat acttatagca 27900

ctgcttggct ttgtgctcta ggaaaggttt taccttttca tagatggcac actatggttc 27960

aaacatgcac acctaatgtt actatcaact gtcaagatcc agctggtggt gcgcttatag 28020

ctaggtgttg gtaccttcat gaaggtcacc aaactgctgc atttagagac gtacttgttg 28080

ttttaaataa acgaacaaat taaaatgtct gataatggac cccaatcaaa ccaacgtagt 28140

gccccccgca ttacatttgg tggacccaca gattcaactg acaataacca gaatggagga 28200

cgcaatgggg caaggccaaa acagcgccga ccccaaggtt tacccaataa tactgcgtct 28260

tggttcacag ctctcactca gcatggcaag gaggaactta gattccctcg aggccagggc 28320

gttccaatca acaccaatag tggtccagat gaccaaattg gctactaccg aagagctacc 28380

cgacgagttc gtggtggtga cggcaaaatg aaagagctca gccccagatg gtacttctat 28440

tacctaggaa ctggcccaga agcttcactt ccctacggcg ctaacaaaga aggcatcgta 28500

tgggttgcaa ctgagggagc cttgaataca cccaaagacc acattggcac ccgcaatcct 28560

aataacaatg ctgccaccgt gctacaactt cctcaaggaa caacattgcc aaaaggcttc 28620

tacgcagagg gaagcagagg cggcagtcaa gcctcttctc gctcctcatc acgtagtcgc 28680

ggtaattcaa gaaattcaac tcctggcagc agtaggggaa attctcctgc tcgaatggct 28740

agcggaggtg gtgaaactgc cctcgcgcta ttgctgctag acagattgaa ccagcttgag 28800

agcaaagttt ctggtaaagg ccaacaacaa caaggccaaa ctgtcactaa gaaatctgct 28860

gctgaggcat ctaaaaagcc tcgccaaaaa cgtactgcca caaaacagta caacgtcact 28920

caagcatttg ggagacgtgg tccagaacaa acccaaggaa atttcgggga ccaagaccta 28980

atcagacaag gaactgatta caaacattgg ccgcaaattg cacaatttgc tccaagtgcc 29040

tctgcattct ttggaatgtc acgcattggc atggaagtca caccttcggg aacatggctg 29100

acttatcatg gagccattaa attggatgac aaagatccac aattcaaaga caacgtcata 29160

ctgctgaaca agcacattga cgcatacaaa acattcccac caacagagcc taaaaaggac 29220

aaaaagaaaa agactgatga agctcagcct ttgccgcaga gacaaaagaa gcagcccact 29280

gtgactcttc ttcctgcggc tgacatggat gatttctcca gacaacttca aaattccatg 29340

agtggagctt ctgctgattc aactcaggca taaacactca tgatgaccac acaaggcaga 29400

tgggctatgt aaacgttttc gcaattccgt ttacgataca tagtctactc ttgtgcagaa 29460

tgaattctcg taactaaaca gcacaagtag gtttagttaa ctttaatctc acatagcaat 29520

ctttaatcaa tgtgtaacat tagggaggac ttgaaagagc caccacattt tcatcgaggc 29580

cacgcggagt acgatcgagg gtacagtgaa taatgctagg gagagctgcc tatatggaag 29640

agccctaatg tgtaaaatta attttagtag tgctatcccc atgtgatttt aatagcttct 29700

taggagaatg acaaaaaaaa aaaaaaaaaa aaaaaa 29736

<210>18

<211>3765

<212>DNA

＜213〉sars coronavirus

<400>18

atgtttattt tcttattatt tcttactctc actagtggta gtgaccttga ccggtgcacc 60

acttttgatg atgttcaagc tcctaattac actcaacata cttcatctat gaggggggtt 120

tactatcctg atgaaatttt tagatcagac actctttatt taactcagga tttatttctt 180

ccattttatt ctaatgttac agggtttcat actattaatc atacgtttgg caaccctgtc 240

atacctttta aggatggtat ttattttgct gccacagaga aatcaaatgt tgtccgtggt 300

tgggtttttg gttctaccat gaacaacaag tcacagtcgg tgattattat taacaattct 360

actaatgttg ttatacgagc atgtaacttt gaattgtgtg acaacccttt ctttgctgtt 420

tctaaaccca tgggtacaca gacacatact atgatattcg ataatgcatt taattgcact 480

ttcgagtaca tatctgatgc cttttcgctt gatgtttcag aaaagtcagg taattttaaa 540

cacttacgag agtttgtgtt taaaaataaa gatgggtttc tctatgttta taagggctat 600

caacctatag atgtagttcg tgatctacct tctggtttta acactttgaa acctattttt 660

aagttgcctc ttggtattaa cattacaaat tttagagcca ttcttacagc cttttcacct 720

gctcaagaca tttggggcac gtcagctgca gcctattttg ttggctattt aaagccaact 780

acatttatgc tcaagtatga tgaaaatggt acaatcacag atgctgttga ttgttctcaa 840

aatccacttg ctgaactcaa atgctctgtt aagagctttg agattgacaa aggaatttac 900

cagacctcta atttcagggt tgttccctca ggagatgttg tgagattccc taatattaca 960

aacttgtgtc cttttggaga ggtttttaat gctactaaat tcccttctgt ctatgcatgg 1020

gagagaaaaa aaatttctaa ttgtgttgct gattactctg tgctctacaa ctcaacattt 1080

ttttcaacct ttaagtgcta tggcgtttct gccactaagt tgaatgatct ttgcttctcc 1140

aatgtctatg cagattcttt tgtagtcaag ggagatgatg taagacaaat agcgccagga 1200

caaactggtg ttattgctga ttataattat aaattgccag atgatttcat gggttgtgtc 1260

cttgcttgga atactaggaa cattgatgct acttcaactg gtaattataa ttataaatat 1320

aggtatctta gacatggcaa gcttaggccc tttgagagag acatatctaa tgtgcctttc 1380

tcccctgatg gcaaaccttg caccccacct gctcttaatt gttattggcc attaaatgat 1440

tatggttttt acaccactac tggcattggc taccaacctt acagagttgt agtactttct 1500

tttgaacttt taaatgcacc ggccacggtt tgtggaccaa aattatccac tgaccttatt 1560

aagaaccagt gtgtcaattt taattttaat ggactcactg gtactggtgt gttaactcct 1620

tcttcaaaga gatttcaacc atttcaacaa tttggccgtg atgtttctga tttcactgat 1680

tccgttcgag atcctaaaac atctgaaata ttagacattt caccttgctc ttttgggggt 1740

gtaagtgtaa ttacacctgg aacaaatgct tcatctgaag ttgctgttct atatcaagat 1800

gttaactgca ctgatgtttc tacagcaatt catgcagatc aactcacacc agcttggcgc 1860

atatattcta ctggaaacaa tgtattccag actcaagcag gctgtcttat aggagctgag 1920

catgtcgaca cttcttatga gtgcgacatt cctattggag ctggcatttg tgctagttac 1980

catacagttt ctttattacg tagtactagc caaaaatcta ttgtggctta tactatgtct 2040

ttaggtgctg atagttcaat tgcttactct aataacacca ttgctatacc tactaacttt 2100

tcaattagca ttactacaga agtaatgcct gtttctatgg ctaaaacctc cgtagattgt 2160

aatatgtaca tctgcggaga ttctactgaa tgtgctaatt tgcttctcca atatggtagc 2220

ttttgcacac aactaaatcg tgcactctca ggtattgctg ctgaacagga tcgcaacaca 2280

cgtgaagtgt tcgctcaagt caaacaaatg tacaaaaccc caactttgaa atattttggt 2340

ggttttaatt tttcacaaat attacctgac cctctaaagc caactaagag gtcttttatt 2400

gaggacttgc tctttaataa ggtgacactc gctgatgctg gcttcatgaa gcaatatggc 2460

gaatgcctag gtgatattaa tgctagagat ctcatttgtg cgcagaagtt caatggactt 2520

acagtgttgc cacctctgct cactgatgat atgattgctg cctacactgc tgctctagtt 2580

agtggtactg ccactgctgg atggacattt ggtgctggcg ctgctcttca aatacctttt 2640

gctatgcaaa tggcatatag gttcaatggc attggagtta cccaaaatgt tctctatgag 2700

aaccaaaaac aaatcgccaa ccaatttaac aaggcgatta gtcaaattca agaatcactt 2760

acaacaacat caactgcatt gggcaagctg caagacgttg ttaaccagaa tgctcaagca 2820

ttaaacacac ttgttaaaca acttagctct aattttggtg caatttcaag tgtgctaaat 2880

gatatccttt cgcgacttga taaagtcgag gcggaggtac aaattgacag gttaattaca 2940

ggcagacttc aaagccttca aacctatgta acacaacaac taatcagggc tgctgaaatc 3000

agggcttctg ctaatcttgc tgctactaaa atgtctgagt gtgttcttgg acaatcaaaa 3060

agagttgact tttgtggaaa gggctaccac cttatgtcct tcccacaagc agccccgcat 3120

ggtgttgtct tcctacatgt cacgtatgtg ccatcccagg agaggaactt caccacagcg 3180

ccagcaattt gtcatgaagg caaagcatac ttccctcgtg aaggtgtttt tgtgtttaat 3240

ggcacttctt ggtttattac acagaggaac ttcttttctc cacaaataat tactacagac 3300

aatacatttg tctcaggaaa ttgtgatgtc gttattggca tcattaacaa cacagtttat 3360

gatcctctgc aacctgagct cgactcattc aaagaagagc tggacaagta cttcaaaaat 3420

catacatcac cagatgttga tcttggcgac atttcaggca ttaacgcttc tgtcgtcaac 3480

attcaaaaag aaattgaccg cctcaatgag gtcgctaaaa atttaaatga atcactcatt 3540

gaccttcaag aattgggaaa atatgagcaa tatattaaat ggccttggta tgtttggctc 3600

ggcttcattg ctggactaat tgccatcgtc atggttacaa tcttgctttg ttgcatgact 3660

agttgttgca gttgcctcaa gggtgcatgc tcttgtggtt cttgctgcaa gtttgatgag 3720

gatgactctg agccagttct caagggtgtc aaattacatt acaca 3765

<210>19

<211>663

<212>DNA

＜213〉sars coronavirus

<400>19

atggcagaca acggtactat taccgttgag gagcttaaac aactcctgga acaatggaac 60

ctagtaatag gtttcctatt cctagcctgg attatgttac tacaatttgc ctattctaat 120

cggaacaggt ttttgtacat aataaagctt gttttcctct ggctcttgtg gccagtaaca 180

cttgcttgtt ttgtgcttgc tgctgtctac agaattaatt gggtgactgg cgggattgcg 240

attgcaatgg cttgtattgt aggcttgatg tggcttagct acttcgttgc ttccttcagg 300

ctgtttgctc gtacccgctc aatgtggtca ttcaacccag aaacaaacat tcttctcaat 360

gtgcctctcc gggggacaat tgtgaccaga ccgctcatgg aaagtgaact tgtcattggt 420

gctgtgatca ttcgtggtca cttgcgaatg gccggacacc ccctagggcg ctgtgacatt 480

aaggacctgc caaaagagat cactgtggct acatcacgaa cgctttctta ttacaaatta 540

ggagcgtcgc agcgtgtagg cactgattca ggttttgctg catacaaccg ctaccgtatt 600

ggaaactata aattaaatac agaccacgcc ggtagcaacg acaatattgc tttgctagta 660

cag 663

<210>20

<211>228

<212>DNA

＜213〉sars coronavirus

<400>20

atgtactcat tcgtttcgga agaaacaggt acgttaatag ttaatagcgt acttcttttt 60

cttgctttcg tggtattctt gctagtcaca ctagccatcc ttactgcgct tcgattgtgt 120

gcgtactgct gcaatattgt taacgtgagt ttagtaaaac caacggttta cgtctactcg 180

cgtgttaaaa atctgaactc ttctgaagga gttcctgatc ttctggtc 228

<210>21

<211>743

<212>DNA

<213>

I type Mahoney strain poliovirus

<400>21

ttaaaacagc tctggggttg tacccacccc agaggcccac gtggcggcta gtactccggt 60

attgcggtac ccttgtacgc ctgttttata ctcccttccc gtaacttaga cgcacaaaac 120

caagttcaat agaagggggt acaaaccagt accaccacga acaagcactt ctgtttcccc 180

ggtgatgtcg tatagactgc ttgcgtggtt gaaagcgacg gatccgttat ccgcttatgt 240

acttcgagaa gcccagtacc acctcggaat cttcgatgcg ttggttagca ctcaacccca 300

gagtgtagct taggctgatg agtctggaca tccctcaccg gtgacggtgt tccaggctgc 360

gttggcggcc tacctatggc taacgcatgg gacgctagtt gtgaacaagg tgtgaagagc 420

ctattgagct acataagaat cctccggccc ctgaatgcgg ctaatcccaa cctcggagca 480

ggtggttcac aaaccagtga ttggcctgtc gtaacgcgca agtccgtggc ggaaccgact 540

actttgggtg tccgtgtttc cttttatttt attgtggctg cttatggtga caatcacaga 600

ttgttatcat aaagcgaatt ggattggcca tccggtgaaa gtgagactca ttatctatct 660

gtttgctgga tccgctccat tgagtgtgtt tactctaagt acaatttcaa cagttatttc 720

aatcagacaa ttgtatcata atg 743

<210>22

<211>397

<212>DNA

＜213〉Rous sarcoma virus

<400>22

cgatgtacgg gccagatata cgcgtatctg aggggactag ggtgtgttta ggcgaaaagc 60

ggggcttcgg ttgtacgcgg ttaggagtcc cctcaggata tagtagtttc gcttttgcat 120

agggaggggg aaatgtagtc ttatgcaata cacttgtagt cttgcaacat ggtaacgatg 180

agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta 240

aggtggtacg atcgtgcctt attaggaagg caacagacag gtctgacatg gattggacga 300

accactgaat tccgcattgc agagataatt gtatttaagt gcctagctcg atacaataaa 360

cgccatttga ccattcacca cattggtgtg cacctcc 397

<210>23

<211>228

<212>DNA

＜213〉simian virus

<400>23

atgtactcat tcgtttcgga agaaacaggt acgttaatag ttaatagcgt acttcttttt 60

cttgctttcg tggtattctt gctagtcaca ctagccatcc ttactgcgct tcgattgtgt 120

gcgtactgct gcaatattgt taacgtgagt ttagtaaaac caacggttta cgtctactcg 180

cgtgttaaaa atctgaactc ttctgaagga gttcctgatc ttctggtc 228

<210>24

<211>32

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>24

aataactgca gcgatgtacg ggccagatat ac 32

<210>25

<211>33

<212>DNA

<213>

Artificial sequence

<220>

＜223〉synthetic Oligonucleolide primers sequence

<400>25

aataagcggc cgcggaggtg cacaccaatg tgg 33

<210>26

<211>2999

<212>DNA

＜213〉artificial sequence

<220>

＜223〉plasmid pVAX1

<400>26

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctacata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc 780

ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 840

gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 900

ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 960

attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 1020

gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt tatggacagc 1080

aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 1140

aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca 1200

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 1260

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 1320

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 1380

cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac 1440

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 1500

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1560

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 1620

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 1680

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 1740

caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 1800

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 1860

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 1920

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 1980

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt 2040

cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacaggtggc 2100

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 2160

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca cgtgctaaaa 2220

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 2280

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2340

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2400

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2460

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2520

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2580

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2640

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2700

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2760

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2820

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2880

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2940

agcaacgcgg cctttttacg gttcctgggc ttttgctggc cttttgctca catgttctt 2999

<210>27

<211>33

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>27

ttattaagct tatgtactca ttcgtttcgg aag 33

<210>28

<211>31

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>28

tattggtacc gaccagaaga tcaggaactc c 31

<210>29

<211>52

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>29

taatatctag agccgccgcc atgtttattt tcttattatt tcttactctc ac 52

<210>30

<211>41

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>30

taatagttta aacttatcat gtgtaatgta atttgacacc c 41

<210>31

<211>13

<212>DNA

<213>

Artificial sequence

<220>

＜223〉KOZAK sequence

<221>misc_feature

<222>7

<223>n＝A or G

<400>31

gccgccncca tgg 13

<210>32

<211>43

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>32

taatagctag cgccgccgcc atggcagaca acggtactat tac 43

<210>33

<211>42

<212>DNA

<213>

Artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>33

gagctgtttt aatcattact gtactagcaa agcaatattgtc 42

<210>34

<211>33

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>34

gctagtacag ttaaaacagc tctggggttg tac 33

<210>35

<211>34

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>35

cgaatgagta cattatgata caattgtctg attg 34

<210>36

<211>32

<212>DNA

<213>

Artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>36

ttgtatcata atgtactcat tcgtttcgga ag 32

<210>37

<211>38

<212>DNA

<213>

Artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>37

tattacttaa gttatcagac cagaagatca ggaactcc 38

<210>38

<211>43

<212>DNA

<213>

Artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>38

taatagctag cgccgccgcc atggcagaca acggtactat tac 43

<210>39

<211>40

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>39

tattagttta aacttatcag accagaagat caggaactcc 40

<210>40

<211>43

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>40

taatagctag cgccgccgcc atggcagaca acggtactat tac 43

<210>41

<211>43

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>41

taatagttta aactcattac tgtactagca aagcaatatt gtc 43

<210>42

<211>33

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>42

ttattgctag catgtactca ttcgtttcgg aag 33

<210>43

<211>40

<212>DNA

＜213〉artificial sequence

<220>

Synthetic Oligonucleolide primers sequence

<223>

<400>43

tattagttta aacttatcag accagaagat caggaactcc 40

<210>44

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence of synthesis method preparation

<400>44

Asp Leu Asp Arg Cys Thr Thr Phe Asp Asp Val G1n Ala Pro Asn

1 5 10 15

<210>45

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence of synthesis method preparation

<400>45

Asp Asp Ser G1u Pro Val Leu Lys G1y Val Lys Leu His Tyr Thr

1 5 10 15

<210>46

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence of synthesis method preparation

<400>46

Met Ala Asp Asn Gly Thr I1e Thr Val Glu G1u Leu Lys Gln Leu

1 5 10 15

<210>47

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence of synthesis method preparation

<400>47

Thr Asp His Ala Gly Ser Asn Asp Asn Ile A1a Leu Leu Val Gln

1 5 10 15

<210>48

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence of synthesis method preparation

<400>48

Met Tyr Ser Phe Val Ser G1u Glu Thr Gly Thr Leu Ile Val Asn

1 5 10 15

<210>49

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide sequence of synthesis method preparation

<400>49

Val Lys Asn Leu Asn Ser Ser Glu G1y Val Pro Asp Leu Leu Val

1 5 10 15

Claims (24)

1. system for preparing the SARS-CoV virus-like particle, described system comprise that one or more express described SARS-CoV E-protein, described SARS-CoV M-protein and the proteinic recombinant vectors of described SARS-CoVS-.

2. the system of claim 1, wherein said SARS-CoV E-protein, described SARS-CoV M-protein and described SARS-CoV S-protein are expressed by single recombinant vectors.

3. the system of claim 1, wherein said SARS-CoV E-protein, described SARS-CoV M-protein and described SARS-CoV S-protein are expressed by a plurality of recombinant vectorss.

4. the system of claim 1, wherein said one or more recombinant vectorss comprise plasmid.

5. the system of claim 1, wherein said one or more recombinant vectorss comprise recombinant virus.

6. the system of claim 5, wherein said recombinant virus is a Measles virus.

7. express SARS-CoV E-protein, SARS-CoV M-protein and the proteinic cell of SARS-CoV S-for one kind, it carries SARS-CoV E-, M-and S-gene.

8. the cell of claim 7, wherein said cell is alive.

9. the cell of claim 8, wherein said cell is a bacterial cell.

10. the cell of claim 9, wherein said cell is its pathogenic bacterial cell that has been weakened.

11. the cell of claim 10, wherein said cell are Salmonellas (Salmonella) cells.

12. a SARS-CoV virus-like particle, it can obtain by each system among the claim 1-6.

13.SARS-CoV the application of virus-like particle in the preparation medicine, described medicine is used for the induce immune response the experimenter.

Described SARS-CoV E-protein, described SARS-CoV M-protein and the proteinic nucleic acid of the described SARS-CoV S-application in the preparation medicine 14. encode, described medicine is used for the induce immune response the experimenter.

15. one or more express the application in the preparation medicine of described SARS-CoV E-protein, described SARS-CoV M-protein and the proteinic recombinant vectors of described SARS-CoV S-, described medicine is used for the induce immune response the experimenter.

16. the application of claim 15, wherein said SARS-CoV E-protein, described SARS-CoVM-protein and described SARS-CoV S-protein are expressed by single recombinant vectors.

17. the application of claim 15, wherein said SARS-CoV E-protein, described SARS-CoVM-protein and described SARS-CoV S-protein are expressed by a plurality of recombinant vectorss.

18. the application of claim 15, wherein said one or more recombinant vectorss comprise plasmid.

19. the application of claim 15, wherein said one or more recombinant vectorss comprise virus.

20. the application of claim 15, wherein said one or more recombinant vectorss comprise prokaryotic vector.

21. the application of claim 15, wherein said experimenter is the people.

22. the application of claim 21, wherein said immunne response is a cellullar immunologic response.

23. the application of claim 21, wherein said immunne response is a humoral immunoresponse(HI).

24. the application of claim 21, wherein said immunne response are body fluid and cellullar immunologic response.

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